Modified plant viruses and methods of use thereof

ABSTRACT

The present invention relates to modulating the nature and/or level of an immune response to a molecule. In particular, the invention relates to effecting an increase in the TH1 immune response to molecules such as, but not limited to, antigens or immunogens. The invention also relates to reducing a TH2 immune response to molecules. More particularly, the invention relates to altering the level of TH1- and TH2-associated immunoglobulins, the level of proliferation of TH1- and TH2-associated cytokines, and the level of proliferation of TH1 and TH2 cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/110,683,filed Apr. 12, 2002, pending, which is a U.S. national entry ofInternational Application No. PCT/US00/28443, filed on Oct. 13, 2000,which claims priority from Great Britain application 9924351.1, filedOct. 14, 1999, now abandoned. The disclosure of each of the previouslyreferenced U.S. patent applications and patents (if applicable)referenced is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to modulating the nature and/or level ofan immune response to a molecule. In particular, the invention relatesto effecting an increase in the TH1 immune response to molecules suchas, but not limited to, antigens or immunogens. The invention alsorelates to reducing a TH2 immune response to molecules. Moreparticularly, the invention relates to altering the level of TH1- andTH2-associated immunoglobulins, the level of proliferation of TH1- andTH2-associated cytokines, and the level of proliferation of TH1 and TH2cells.

BACKGROUND OF THE INVENTION

The priming of the TH1, rather than TH2, subset of CD4+ T cells is ofprimary importance in vaccine design. This is because TH1 cells produceIL-2, IFN-γ, and TNF-β cytokines that mediate macrophage and cytotoxic Tcell activation (CTL), and are the principal effectors of cell-mediatedimmunity against intracellular microbes and of delayed typehypersensitivity (DTH) reactions. IFN-γ also induces B cell isotypeclass-switching to the principal effector isotype of mouse IgG. IgG2a,and to a lesser extent IgG2b, enhances antibody-dependent cell mediatedcytotoxicity (ADCC), and strongly binds Clq of the classical complementpathway which opsonizes cells or antibody clusters for phagocytosis. Forexample, as a result of these effector functions in mouse, IgG2a hasbeen found to better protect mice against virus infections (Ishizaka etal. (1995) J. Infect. Dis. 172:1108), murine tumors (Kaminski et al.(1986) J. Immunol. 136:1123), and parasites (Wechsler et al. (1986) J.Immunol. 137:2968), and to enhance bacterial clearance (Zigterman et al.(1989) Infect. Immun. 57:2712).

In contrast, TH2 cells produce IL-4, IL-5, IL-10, and IL-13 which havethe undesirable effect of suppressing cell mediated immunity [Mossman etal. (1986) J. Immunol. 136:2248]. Furthermore, IL-4 induces B cells toproduce both an IgG subclass which poorly fixes complement and does notmediate ADCC, as well as IgE which binds to mast cells and basophils.

The prior art has attempted to improve vaccine design by directing thedevelopment of TH1 cells, while recognizing that the priming of TH cellsubsets is affected by the strain of animal used (Hocart et al. (1989)J. Gen. Virol. 70:2439), the identity of the antigen, the route ofantigen delivery (Hocart et al. (1989) J. Gen. Virol. 70:809), and theimmunization regimen (Brett et al. (1993) Immunology 80:306).

One approach of the prior art to generate antigen-specific TH1 responseshas been through the use of the oxidative/reductive conjugation ofmannan to antigen (Apostopoulos et al. (1995) Proc. Natl. Acad. Sci. USA92:10128-10132) or the conjugation of proteins to bacterial proteins(Jahn-Schmid et al. (1997) Intl. Immunol. 9:1867). However coupling tocommonly used carriers such as KLH and tetanus toxoid is oftenunsuccessful at increasing the IgG2a:IgG1 ratio.

Other methods of inducing TH1 responses include the use ofimmunomodulatory agents such as extraneous adjuvants (for example:ISCOMS, QS-21, Quil A, etc.). However, alum is the only adjuvant whichis currently approved for use in humans and is known to favorundesirable TH2-type responses rather than the more desirable TH1 typeresponse.

Other immunomodulatory agents which have been used by the prior artinclude CpG oligodeoxyribonucleotides (Chu et al. (1997) J. Exp. Med.186:1623) or cytokines such as interleukin-12 (IL-12, Scott et al (1997)Seminl. Immunol. 9:285), which may be added to immunogenic compositionsleading to the production of high levels of IgG_(2a) directed againstsoluble protein antigens. However, the cytokines' short half-life andconsiderable cost make utilizing them both technically and commerciallyunattractive in large-scale vaccination.

Yet another approach has been to use live animal virus vaccines togenerate predominantly virus-specific IgG_(2a) in mice (Hocart et al(1989) J. Gen. Virol. 70:809; Coutelier et al. (1987) J. Exp. Med.165:64; Nguyen et al. (1994) J. Immunol. 152:478; Brubaker et al. (1996)J. Immunol. 157:1598). This approach has several disadvantages. First,live virus vaccines do not consistently result in a TH1 type immuneresponse, since some live viruses favor production of otherimmunoglobulin isotypes characteristic of alternate T helper pathways(Coutelier et al. (1987) J. Exp. Med. 165:64; Perez-Filgueira et al.(1995) Vaccine 13:953). In addition, such animal virus vaccines areproduced from viruses which are grown in cell culture systems that areexpensive to design and run.

Moreover, the animal virus used as the vector is often a virus to whichthe animal may already have been exposed, and the animal may already beproducing antibodies to the vector. The vector may therefore bedestroyed by the immune system before the incorporated antigenic site ofthe second virus induces an immune response. Additionally, the compositeanimal virus approach involves genetic manipulation of live,animal-infecting viruses, with the risk that mutations may give rise tonovel forms of the virus with altered infectivity, antigenicity and/orpathogenicity. Indeed, there are safety concerns over the use of liveanimal viral vaccines (World Health Organization, 1989). Furthermore,the safety concerns over the use of live animal viral vaccines are notovercome by using inactive animal viruses since inactive animal virusesgenerally do not favor IgG_(2a) production. Indeed, it is thought thatthe infection process typified by live viruses per se generates IFN-γleading to a predominance of TH1 response immunoglobulins (Nguyen et al.(1994) J. Immunol. 152:478).

While another approach has involved using live bacterial vectors and DNAimmunization which favor the generation of TH1 responses to expressedpeptides, this approach is however often dependent on, and sensitive to,either the route of delivery or adjuvant used. Moreover, safety concernsover the use of live bacterial vaccines and DNA vaccines (World HealthOrganization, 1989) further limit their clinical application.

Thus, there is a need for compositions and methods of generatingTH1-type responses. Preferably, the generation of TH1-type responses bythese compositions and methods is unaffected by the genetic backgroundof the host animal, the identity of the antigen, the route of antigendelivery, and the immunization regimen.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions which areeffective in modulating the nature and/or level of an immune response toa molecule exemplified by, but not limited to, an antigen or immunogen.In particular, the invention provides methods and means for effecting aTH1 bias in the immune response to molecules such as antigens orimmunogens, and/or reducing a TH2 bias in the immune response to suchmolecules. More particularly, the invention provides methods and meansfor increasing a TH1 immune response which is directed against moleculesthat otherwise generally stimulate a TH2-type response. The inventionfurther provides compositions and methods to reduce a TH2 immuneresponse to molecules. Additionally provided herein are compositions andmethods for altering (that is, increasing or decreasing) the level ofTH1- and TH2-associated immunoglobulins, the level of proliferation ofTH1- and TH2-associated cytokines, and the level of proliferation of TH1and TH2 cells.

More particularly, the invention provides a method of altering the levelof TH1-associated immunoglobulin in an animal, comprising: a) providing:i) aplant virus containing a heterologous peptide; and ii) a hostanimal; b) administering the plant virus to the host animal to generatea treated animal; c) testing for an increase in the level ofTH1-associated immunoglobulin in the treated animal relative to thelevel of TH1-associated immunoglobulin in a control animal. In oneembodiment, the method further comprises d) observing an increase in thelevel of TH1-associated immunoglobulin in the treated animal. Withoutintending to limit the invention to any particular route ofadministration, in another embodiment, the administering is selectedfrom intranasal, oral, parenteral, subcutaneous, intrathecal,intravenous, intraperitoneal, and intramuscular administration. Also,without limitation of the invention to the type or source ofcompositions included in administration of the invention's viruses, inyet another embodiment, the administering further comprisesadministering a composition selected from immune adjuvant, cytokine, andpharmaceutical excipient. In a further embodiment, the administeringresults in reducing symptoms associated with exposure of the host animalto the heterologous peptide.

Although it is not intended that the invention be limited to anyparticular type of host animal, in yet another embodiment, the hostanimal is a mammal. It is not intended that the invention be limited toany particular mammal. However, in a preferred embodiment, the mammal isselected from mouse and human. In a more preferred embodiment, the hostanimal is mouse, and the TH1-associated immunoglobulin is selected fromIgG2a and IgG2b. In a yet more preferred embodiment, the TH1-associatedimmunoglobulin is IgG2a. In another preferred embodiment, the hostanimal is human, and the TH1-associated immunoglobulin is selected fromIgG1 and IgG3.

Without restricting the specificity of the TH1-associatedimmunoglobulin, in an alternative embodiment, the TH1-associatedimmunoglobulin is selected from immunoglobulin specific for theheterologous peptide, and immunoglobulin specific for the plant virus.In another alternative embodiment, the plant virus is an RNA virus. In afurther alternative embodiment, the plant virus is an icosahedral plantvirus selected from Comoviruses, Tombusviruses, Sobemoviruses, andNepoviruses. In a preferred embodiment, the plant virus is a Comovirus.In a more preferred embodiment, the Comovirus is cowpea mosaic virus(CPMV).

While the invention is not limited to any particular source or type ofheterologous peptide, in an additional alternative embodiment, theheterologous peptide is selected from the β-subunit of the humanchorionic gonadotrophin, human membrane bound IgE, human mutantepidermal growth factor receptor variant III, canine parvovirus, apeptide hormone, a gonadotrophin releasing hormone, an allergen, apeptide derived from a cancer cell, and a peptide derived from an animalpathogen.

The invention is not intended to be limited to the location on the virusat which the heterologous peptide is expressed. Nonetheless, in yetanother alternative embodiment, the heterologous peptide is expressed atan exposed portion of the coat protein of the plant virus. In apreferred embodiment, the coat protein has a beta-barrel structure andthe heterologous peptide is inserted in a loop between individualstrands of the beta sheet of the beta barrel structure. In a morepreferred embodiment, the heterologous peptide is inserted in the βB-βPCloop of the plant virus. In another preferred embodiment, theheterologous peptide is inserted between alanine 22 and proline 23 ofthe small coat protein (VP-S) of cowpea mosaic virus. In anotherembodiment, a nucleic acid sequence encoding the heterologous peptide isinserted in the plant virus genome at a site which is free from directnucleotide sequence repeats flanking the nucleic acid sequence.

It is not contemplated that the invention be restricted to the type ofheterologous peptide. However, in yet another embodiment, theheterologous peptide is antigenic. In a preferred embodiment, theheterologous peptide is derived from an animal virus. In a morepreferred embodiment, the animal virus is selected from foot-and-mouthdisease virus, human immune deficiency virus, human rhinovirus, canineparvovirus. In an alternative preferred embodiment, the heterologouspeptide is derived from a composition selected from an animal pathogen,a hormone, and cytokine. In a more preferred embodiment, the animalpathogen is selected from a virus, bacterium, protozoan, nematode, andfungus. In yet a further embodiment, the heterologous peptide isimmunogenic.

The invention additionally provides a method of altering the level ofproliferation of TH1 cells in an animal, comprising: a) providing: i) aplant virus containing a heterologous peptide; and ii) a host animal; b)administering the plant virus to the host animal to generate a treatedanimal; and c) testing for an increase in the level of proliferation ofTH1 cells from the treated animal relative to the level of proliferationof TH1 cells from a control animal. In one embodiment, the methodfurther comprises d) observing an increase in the proliferation level ofTH1 cells from the treated animal relative to the proliferation level ofTH1 cells from a control animal. In another embodiment, theadministering results in reducing symptoms associated with exposure ofthe host animal to the heterologous peptide.

Also provided herein is a method of altering the level of aTH1-associated cytokine in an animal, comprising: a) providing: i) aplant virus containing a heterologous peptide; and ii) a host animal;and b) administering the plant virus to the host animal to generate atreated animal; and c) testing for an increase in the level of thecytokine produced by T cells from the treated animal relative to thelevel of the cytokine produced by T cells from a control animal. In oneembodiment, the method further comprises d) observing an increase in thelevel of the cytokine produced by T cells from the treated animalrelative to the level of the cytokine produced by T cells from a controlanimal. In another embodiment, the administering results in reducingsymptoms associated with exposure of the host animal to the heterologouspeptide. In yet another embodiment, the cytokine is selected from IL-2,TNF-β and IFN-γ. In a preferred embodiment, the cytokine is INF-γ. In afurther embodiment, the level of a TH2-associated cytokine in thetreated animal is the same as the level of the second cytokine in thehost animal. In a preferred embodiment, the second cytokine is selectedfrom IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. In a more preferredembodiment, the second cytokine is IL-4. In an additional embodiment,the level of a TH2-associated cytokine in the treated animal is reducedrelative to the level of the second cytokine in the host animal.

The invention additionally provides a method of increasing the level ofa TH1-type immune response to a molecule of interest in an animalcomprising: a) providing: i) the molecule of interest; ii) a plant virusexpressing a heterologous peptide capable of conjugating to the moleculeof interest; and iii) a host animal; b) conjugating the molecule ofinterest to the heterologous peptide to generate a conjugate; and c)administering the conjugate to the host animal to generate a treatedanimal under conditions such that the level of TH1-type immune responseto the molecule of interest in the treated animal is increased relativeto the level of TH1-type immune response to the molecule of interest ina control animal. In one embodiment, the method further comprises d)testing for an increase in the level of TH1-type immune response to themolecule of interest in the treated animal relative to the level ofTH1-type immune response to the molecule of interest in a controlanimal. In a preferred embodiment, the method further comprises e)observing an increase in the level of TH1-type immune response to themolecule of interest in the treated animal relative to the level ofTH1-type immune response to the molecule of interest in a controlanimal.

While not limiting the nature or extent of the increased level ofTH1-type response, in another preferred embodiment, the increased levelof TH1 type immune response is selected from (a) increased level ofTH1-associated immunoglobulin in the treated animal relative to thelevel of TH1-associated immunoglobulin in a control animal, (b)increased level of proliferation of TH1 cells from the treated animalrelative to the level of proliferation of TH1 cells from a controlanimal, and (c) increased level of TH1-associated cytokine in thetreated animal relative to the level of TH1-associated cytokine in acontrol animal. In another embodiment, the TH1-associated cytokine isselected from IL-2, TNF-β and IFN-γ. In a more preferred embodiment, thecytokine is INF-γ. In yet another embodiment, the administering resultsin reducing symptoms associated with exposure of the host animal to themolecule of interest. In an alternative embodiment, the molecule ofinterest comprises a peptide, polysaccharide, nucleic acid, or lipid. Ina preferred embodiment, the molecule of interest is derived from asource selected from an animal pathogen, an allergen, and a cancer cell.

It is not intended that the invention be limited to the type or naturaof virus. However, in another alternative embodiment, the plant virus isan icosahedral plant virus selected from Comoviruses, Tombusviruses,Sobemoviruses, and Nepoviruses.

In a preferred embodiment, the plant virus is a Comovirus. In a morepreferred embodiment, the Comovirus is cowpea mosaic virus (CPMV).

Although the location of introduction of the heterologous peptide intothe virus is not intended to be limited, in a yet more preferredembodiment, the heterologous peptide is inserted between alanine 22 andproline 23 of the small coat protein (VP-S) of cowpea mosaic virus. In afurther alternative embodiment, the heterologous peptide is expressed atan exposed portion of the coat protein of the plant virus.

The invention is not limited to the nature or type of heterologouspeptide. Nonetheless, in an additional alternative embodiment, theheterologous peptide comprises one or more charged amino acids selectedfrom negatively charged amino acids and positively charged amino acids,wherein the negative charge on the negatively charged amino acids isbalanced by the positive charge on the positively charged amino acids.In a preferred embodiment, the negatively charged amino acids areselected from aspartic acid, glutamic acid, and cysteine. In anotherpreferred embodiment, the positively charged amino acids are selectedfrom lysine, arginine, and histidine. In yet another preferredembodiment, the heterologous peptide comprises a sequence of contiguouscharged amino acids selected from a first sequence consisting ofcontiguous negatively charged amino acids and a second sequenceconsisting of contiguous positively charged amino acids. In a morepreferred embodiment, the sequence of contiguous charged amino acidsoccurs in the heterologous peptide as a repeating sequence. In anothermore preferred embodiment, the heterologous sequence comprises the firstand second sequences. In a yet more preferred embodiment, the firstsequence is contiguous with the second sequence. In a particularlypreferred embodiment, the contiguous first and second sequences occur inthe heterologous peptide as a repeating sequence. In a further preferredembodiment, the heterologous peptide comprises non-contiguous negativelycharged amino acids and non-contiguous positively charged amino acids.In a more preferred embodiment, the heterologous peptide furthercomprises a sequence of contiguous charged amino acids selected from afirst sequence consisting of contiguous negatively charged amino acids,and a second sequence consisting of contiguous positively charged aminoacids. In a yet more preferred embodiment, the heterologous peptidecomprises the first sequence. In an additionally preferred embodiment,the first sequence of contiguous negatively charged amino acids has thegeneral formula Asp-Glu_(n)-Gly-Lys_(2n)-Asp-Glu_(n) listed as SEQ IDNO:16, where n is an integer of from 1 to 40. In a particularlypreferred embodiment, the first sequence is the amino acid sequenceAsp-Glu-Gly-Lys-Gly-Lys-Gly-Lys-Gly-Lys-Asp-Glu listed as SEQ ID NO:20.

Without limiting the route or mode of administration, in a furtherembodiment, the administering is selected from intranasal, oral,parenteral, subcutaneous, intrathecal, intravenous, intraperitoneal, andintramuscular administration. In an additional embodiment, theadministering further comprises administering a composition selectedfrom immune adjuvant, cytokine, and pharmaceutical excipient.

While the type of animal is not limited, the invention contemplatesthat, in yet another embodiment, the host animal is a mammal. In apreferred embodiment, the mammal is selected from mouse and human. In analternative embodiment, the conjugate is immunogenic.

Also provided by the invention is a method of reducing the level ofTH2-type immune response to a molecule of interest, comprising: a)providing: i) the molecule of interest; ii) a plant virus; and iii) ahost animal; b) conjugating the molecule of interest to the plant virusto generate a conjugate; and c) administering the conjugate to the hostanimal to generate a treated animal under conditions such that the levelof TH2-type immune response to the molecule of interest in the treatedanimal is reduced relative to the level of TH2-type immune response tothe molecule of interest in a control animal. In one embodiment, themethod further comprises d) testing for a reduction in the level ofTH2-type immune response to the molecule of interest in the treatedanimal relative to the level of TH2-type immune response to the moleculeof interest in a control animal. In a preferred embodiment, the methodfurther comprises e) observing a reduction in the level of TH2-typeimmune response to the molecule of interest in the treated animalrelative to the level of TH2-type immune response to the molecule ofinterest in a control animal. In another embodiment, the administeringresults in reducing symptoms associated with exposure of the host animalto the molecule of interest. In an alternative embodiment, the reducedlevel of TH2 type immune response is selected from (a) reduced level ofTH2-associated immunoglobulin in the treated animal relative to thelevel of TH2-associated immunoglobulin in a control animal, (b) reducedlevel of proliferation of TH2 cells from the treated animal relative tothe level of proliferation of TH2 cells from a control animal, and (c)reduced level of TH2-associated cytokine in the treated animal relativeto the level of TH2-associated cytokine in a control animal. In apreferred embodiment, the TH2-associated cytokine is selected from IL-4,IL-5, IL-6, IL-9, IL-10, and IL-13. In a more preferred embodiment, theTH2-associated cytokine is IL-4. In another alternative embodiment, themolecule of interest comprises a peptide, polysaccharide, nucleic acid,or lipid. In an additional alternative embodiment, the molecule ofinterest is selected from an antigen and adjuvant. In a preferredembodiment, the antigen is a peptide. In yet another alternativeembodiment, the host animal is mouse, and the TH2-associatedimmunoglobulin is selected from IgG1 and IgG3. In an additionalembodiment, the host animal is human, and the TH2-associatedimmunoglobulin is IgG2.

The invention also provides a method of reducing the level of an extantTH2-type immune response to a molecule of interest, comprising: a)providing: i) the molecule of interest; ii) a plant virus; and iii) ahost animal exhibiting a TH2-type immune response to the molecule ofinterest; b) conjugating the molecule of interest to the plant virus togenerate a conjugate; and c) administering the conjugate to the hostanimal to generate a treated animal under conditions such that the levelof TH2-type immune response in the treated animal is reduced relative tothe level of TH2-type immune response in the host animal. In oneembodiment, the method further comprises d) testing for a reduction inthe level of TH2-type immune response to the molecule of interest in thetreated animal relative to the level of TH2-type immune response to themolecule of interest in a control animal. In a preferred embodiment, themethod further comprises e) observing a reduction in the level ofTH2-type immune response to the molecule of interest in the treatedanimal relative to the level of TH2-type immune response to the moleculeof interest in a control animal. In another embodiment, the TH2-typeresponse in the host animal is dominant.

The invention also provides a process of increasing the level of aTH1-type immune response to a molecule of interest in an animal,comprising: a) providing: i) said molecule of interest; ii) a plantvirus expressing a heterologous peptide capable of conjugating to saidmolecule of interest; and iii) a host animal; b) conjugating saidmolecule of interest to said heterologous peptide to generate aconjugate; c) administering said conjugate to said host animal togenerate a treated animal under conditions such that the level ofTH1-type immune response to said molecule of interest in said treatedanimal is increased relative to the level of TH1-type immune response tosaid molecule of interest in a control animal; d) optionally testing foran increase in the level of TH1-type immune response to said molecule ofinterest in said treated animal relative to the level of TH1-type immuneresponse to said molecule of interest in a control animal; and e)optionally observing an increase in the level of TH1-type immuneresponse to said molecule of interest in said treated animal relative tothe level of TH1-type immune response to said molecule of interest in acontrol animal. In one embodiment, the increased level of TH1-typeimmune response is selected from (a) increased level of TH1-associatedimmunoglobulin in said treated animal relative to the level ofTH1-associated immunoglobulin in a control animal, (b) increased levelof proliferation of TH1 cells from said treated animal relative to thelevel of proliferation of TH1 cells from a control animal, and (c)increased level of TH1-associated cytokine in said treated animalrelative to the level of TH1-associated cytokine in a control animal. Inanother embodiment, the TH1-associated cytokine is selected from IL-2,TNF-β and IFN-γ. In yet another embodiment, the administering results inreducing symptoms associated with exposure of said host animal to saidmolecule of interest. In a further embodiment, the molecule of interestcomprises a peptide, polysaccharide, nucleic acid, or lipid. In analternative embodiment, the molecule of interest is derived from asource selected from an animal pathogen, an allergen, and a cancer cell.In yet another embodiment, the plant virus is an icosahedral plant virusselected from Comoviruses, Tombusviruses, Sobemoviruses, andNepoviruses. In an alternative embodiment, the plant virus is aComovirus. In a more preferred embodiment, the Comovirus is cowpeamosaic virus (CPMV). In yet another embodiment, the heterologous peptideis expressed at an exposed portion of the coat protein of said plantvirus. In an alternative embodiment, the heterologous peptide comprisesone or more charged amino acids selected from negatively charged aminoacids and positively charged amino acids, wherein the negative charge onsaid negatively charged amino acids is balanced by the positive chargeon said positively charged amino acids. In another embodiment, thenegatively charged amino acids are selected from aspartic acid, glutamicacid, and cysteine, and said positively charged amino acids are selectedfrom lysine, arginine, and histidine. In another embodiment, theheterologous peptide comprises a sequence of contiguous charged aminoacids selected from a first sequence consisting of contiguous negativelycharged amino acids and a second sequence consisting of contiguouspositively charged amino acids. In yet another embodiment, the sequenceof contiguous charged amino acids occurs in said heterologous peptide asa repeating sequence. In a further embodiment, the heterologous sequencecomprises said first and second sequences, and wherein said firstsequence is contiguous with said second sequence. In yet a furtherembodiment, the contiguous first and second sequences occur in saidheterologous peptide as a repeating sequence. In still a furtherembodiment, the heterologous peptide comprises non-contiguous negativelycharged amino acids and non-contiguous positively charged amino acids,and wherein said heterologous peptide further comprises a sequence ofcontiguous charged amino acids selected from a first sequence consistingof contiguous negatively charged amino acids, and a second sequenceconsisting of contiguous positively charged amino acids. In anotherembodiment, the first sequence of contiguous negatively charged aminoacids has the general formula Asp-Glu_(n)-Gly-Lys_(2n)-Asp-Glu_(n)listed as SEQ ID NO: 16, where n is an integer of from 1 to 40. In stillanother embodiment, the administering is selected from intranasal, oral,parenteral, subcutaneous, intrathecal, intravenous, intraperitoneal, andintramuscular administration. In yet another embodiment, theadministering further comprises administering a composition selectedfrom immune adjuvant, cytokine, and pharmaceutical excipient. In afurther embodiment, the host animal is a mammal. In another alternativeembodiment, the mammal is selected from mouse and human. In onepreferred embodiment, the conjugate is immunogenic.

The invention also provides a method for stimulating a predominantlyTH1-type peptide-specific immune response comprising the conjugation ofan antigen to a plant virus, and administering the resultant immunogeniccomplex to an animal. Preferably, the plant virus is an icosahedralplant virus. More preferably, the plant virus is a comovirus. Yet morepreferably, the plant virus is cowpea mosaic virus. In one embodiment,the antigen is a foreign peptide which is expressed as an insertionpolypeptide encoded by a recombinant structural gene of the plant virus.Preferably, the antigen is expressed as a fusion polypeptide encoded bya recombinant coat protein structural gene. In yet another embodiment,the immunogenic complex is administered in the presence of apharmaceutically acceptable excipient. In a further embodiment, the modeof administration of the immunogenic complex is selected from intranasalinoculation, oral inoculation, parenteral inoculation, and subcutaneousinoculation. In yet another embodiment, the immunogenic complex isadministered in the presence of an immunomodulatory agent. Preferably,the immunomodulatory agent is an immune adjuvant. More preferably, theimmune adjuvant is selected from the group comprising: cholera toxin(CT) and mutants thereof, heat labile enterotoxin (LT) and mutantsthereof, Quil A (QS-21), the R1B1 adjuvant and ISCOMs. In an alternativepreferred embodiment, the immunomodulatory agent is a cytokine,preferably a cytokine which can be secreted from a CD4+ TH1 lymphocyteof an animal. More preferably, the cytokine is selected from the groupof interleukin-2, interferon-γ, and tumor necrosis factor-β. In anadditional embodiment, the immunogenic complex is administered by asubcutaneous route at one site in a single dose, by a subcutaneous routeat more than one site in a single contemporaneous dose, by a parenteralroute at one site in a single dose, by a parenteral route at more thanone site in a single contemporaneous dose, by a subcutaneous route atone site in more than one dose, by a parenteral route at one site inmore than one dose, by a subcutaneous route at more than one site inmore than one dose, and/or by a parenteral route at more than one sitein more than one dose.

Also provided by the invention is a chimeric virus particle, which isuseful in any of the above-described methods, and in which a reactivepeptide capable of having chemically conjugated to it a proteinaceous ornon-proteinaceous molecule, is encoded within a structural gene of thechimeric plant virus genome. In one embodiment, the chemically reactivepeptide is inserted into a coat protein of the chimeric virus particle.In another embodiment, the insertion of the chemically reactive peptideis between alanine 22 and proline 23 of the small coat protein (VP-S) ofcowpea mosaic virus. In yet another embodiment, the positive charges onthe reactive amino acid residues (X) are balanced by the inclusion of acorresponding number of negatively charged amino acid residues (Y)within the reactive peptide, conforming to a general formula X(n)Y(n),where n is an integer of between 1 and 40. In another embodiment, theinserted chemically reactive peptide has an amino acid sequence of thegeneral formula DE(n)GK(n)DE(n) listed as SEQ ID NO:16, where n is aninteger of between 1 and 40. In a preferred embodiment, the insertedchemically reactive peptide has the amino acid sequence DEGKGKGKGKDE(SEQ ID NO:20, also listed as SEQ ID NO:29). In a further embodiment,the antigen is a carbohydrate moiety conjugated via a chemical bond to areactive peptide expressed as an inserted fusion within a structuralprotein of a chimeric plant virus.

The invention also provides a method of by-passing a TH2-type immunereaction favored by the inherent immune stimulatory characteristics ofan antigen comprising conjugating the antigen to a plant virus andadministering the resultant immunogenic complex to an animal.

Also disclosed herein is a method of by-passing a TH2-type immunereaction favored by the inherent immune stimulatory characteristics of apeptide comprising expressing the peptide as a fusion polypeptideencoded by the recombinant genome of a plant virus and administering theresultant immunogenic complex to an animal.

The invention additionally provides a method of by-passing a TH2-typeimmune reaction favored by the genetic constitution of an animal to anantigen comprising conjugating the antigen to a plant virus andadministering the resultant immunogenic complex to the animal.

Moreover, the invention provides a method of by-passing a TH2-typeimmune reaction favored by the genetic constitution of an animal to apeptide comprising expressing the peptide as a fusion polypeptideencoded by the recombinant genome of a plant virus and administering theresultant immunogenic complex to an animal.

Also provided by the invention is a method of by-passing a TH2-typeimmune reaction favored by inherent immune stimulatory characteristicsof an adjuvant present in an immunogenic complex containing an antigencomprising conjugating the antigen to a plant virus and administeringthe resultant immunogenic complex to an animal.

The invention additionally provides a method of treating an animal foran infectious disease by the stimulation of a TH1-biased immune responsecomprising conjugating an antigen associated with the infectious agentto a plant virus and administering the resultant immunogenic complex tothe animal.

Also, the invention provides a method of treating an animal for anallergy by the stimulation of a TH1-biased immune response comprisingconjugating an antigen derived from the cognate allergen associated withthe allergy to a plant virus and administering the resultant immunogeniccomplex to the animal.

In addition, the invention discloses a method of treating an animalsuffering from cancer by the stimulation of a TH1-biased immune responsecomprising conjugating an antigen associated with the cancer to a plantvirus and administering the resultant immunogenic complex to the animal.

Furthermore, provided herein is a method for inducing a shift in anextant TH2-type immune response to an antigen present in one immunogeniccomplex used to prime an immune response in an animal which comprisesinoculating an animal with an immunogenic complex containing thatantigen conjugated to a plant virus particle in a secondary orsubsequent booster dose.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The terms “antigen” and “antigenic” refer to any substance that isspecifically recognized by antibody or a T cell receptor. Antigenscontain one or more epitopes (also referred to as “antigenicdeterminants”). An “epitope” is a structure on an antigen whichinteracts with the binding site of an antibody or T cell receptor as aresult of molecular complementarity. An epitope may compete with theintact antigen, from which it is derived, for binding to an antibody.Generally, secreted antibodies and their corresponding membrane-boundforms are capable of recognizing a wide variety of substances asantigens, whereas T cell receptors are capable of recognizing onlyfragments of proteins which are complexed with MHC molecules on cellsurfaces. Antigens recognized by immunoglobulin receptors on B cells aresubdivided into three categories: T-cell dependent antigens, type 1 Tcell-independent antigens; and type 2 T cell-independent antigens. Anantigen may contain one or more of the following molecules: a peptide,polysaccharide, nucleic acid sequence, and lipid.

The terms “immunogen,” “immunogenic,” and “immunologically active” referto any substance that is capable of inducing a specific humoral orcell-mediated immune response. By definition, an immunogen must containat least one epitope, and generally contains several epitopes.Immunogens are exemplified by, but not restricted to molecules whichcontain a peptide, polysaccharide, nucleic acid sequence, and/or lipid.Complexes of peptides with lipids, polysaccharides, or with nucleic acidsequences are also contemplated, including (without limitation)glycopeptide, lipopeptide, glycolipid, etc. These complexes areparticularly useful immunogens where smaller molecules with few epitopesdo not stimulate a satisfactory immune response by themselves.

The term “allergen” refers to an antigen or immunogen which induces oneor more hypersensitivity (allergic) reactions, exemplified, but notlimited to the production of IgE antibodies. Allergens include, but arenot restricted to, pollens of ragweed, grasses, or trees, or those offungi, animal danders, house dust, or foods. Individuals exposed to anallergen generally, though not necessarily, develop hives or themanifestations of hay fever or asthma.

The term “adjuvant” as used herein refers to any compound which, wheninjected together with an antigen, non-specifically enhances the immuneresponse to that antigen.

The term “excipient” refers herein to any inert substance (for example,gum arabic, syrup, lanolin, starch, etc.) that forms a vehicle fordelivery of an antigen. The term excipient includes substances which, inthe presence of sufficient liquid, impart to a composition the adhesivequality needed for the preparation of pills or tablets.

The terms “antibody” and “immunoglobulin” are interchangeably used torefer to a glycoprotein evoked in an animal by an immunogen. An antibodydemonstrates specificity to the immunogen, or, more specifically, to oneor more epitopes contained in the immunogen.

Native antibody comprises at least two light polypeptide chains and atleast two heavy polypeptide chains. Antibodies may be polyclonal ormonoclonal. The term “polyclonal antibody” refers to immunoglobulinproduced from more than a single clone of plasma cells; in contrast“monoclonal antibody” refers to immunoglobulin produced from a singleclone of plasma cells.

The term “isotype” or “class” when made in reference to an antibodyrefers to an antibody class that is encoded by a particular type ofheavy chain constant region gene. Thus, different antibody isotypesdiffer in the amino acid sequence of the heavy chain constant (CH)region. Several antibody isotypes are known, including IgA, IgD, IgG,IgE, and IgM. Thus, IgG possesses γ heavy chain constant domain (Cy);IgM possesses μ heavy chain constant domain (Cp); IgA possesses ∝ heavychain constant domain (C∝); IgD possesses δ heavy chain constant domain(Cδ); and IgE possesses ε heavy chain constant domain (Cε). Differentantibody classes exhibit different effector functions and displaydifferent tissue localization. Each antibody class can be expressed as amembrane (m) or a secreted (s) form which differ in sequence at thecarboxyl terminus of the heavy chain. Most antigens elicit prompt serumexpression of IgM, followed later by a secondary isotype switchingresponse in which products of downstream heavy chain genes, such as Cγ1and Cγ2a predominate. The antibody isotype which predominates generallydepends on the type of antigen (for example, polypeptide,polysaccharide, etc.) used to elicit production of the antibody.

The terms “subtype” and “subclass” when made in reference to an antibodyisotype, interchangeably refer to antibodies within an isotype whichcontain variation in the heavy chain structure. For example, the humanIgG class contains the four isotypes IgG1, IgG2, IgG3, and IgG4, whilethe mouse IgG isotype contains the four subtypes IgG1, IgG2a, IgG2b,IgG3. Human IgA isotype contains IgA1 and IgA2 subtypes. The genescoding for the constant heavy chain have been mapped in mouse and arelocated on chromosome 12 in the order (from the 5′ end)Cμ-Cδ-Cγ3-Cγ1-Cγ2b-Cγ2a-Cε-C∝ corresponding to isotypes IgM, IgD, IgG3,IgG1, IgG2b, IgG2a, IgE and IgA.

The term “isotype switching” refers to the phenomenon by which oneantibody isotype (for example, IgM) changes to another antibody isotype(for example, IgG). Similarly, the term “subtype switching” refers tothe phenomenon by which an antibody subtype (for example, IgG1) changesto another subtype (for example, IgG2a) of the same antibody isotype.

The term “effector functions” as used herein in reference to an antibodyrefer to non antigen-binding activities which are mediated by theantibody molecules, and which are executed through the constant heavychain region of the molecule. Effector functions are exemplified byreceptor-binding on immune cells, complement fixation, etc. Effectorfunctions facilitate the removal of antigen from the body by means ofcomplement-mediated lysis or phagocytosis.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and an antigen means thatthe interaction preferably shows a preference for, and more preferablyis dependent upon, the presence of a particular structure (that is, theantigenic determinant or epitope) on the antigen; in other words theantibody is recognizing and binding to a specific antigen structure(including related structures) rather than to antigens in general. Forexample, if an antibody is specific for epitope “A,” the presence of anantigen containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein the term “immunogenically-effective amount” refers tothat amount of an immunogen required to invoke the production ofantibodies in a host upon vaccination. It is preferred, though notrequired, that the immunologically-effective amount is a “protective”amount. The terms “protective” and “therapeutic” amount of an immunogenrefer to that amount of the immunogen which diminishes one or moreundesirable symptoms that are associated with exposure of the hostanimal to the immunogen. The terms to “diminish” and “reduce” symptomsas used herein in reference to the effect of a particular composition orof a particular method is meant to reduce, delay, or eliminate one ormore symptoms as compared to the symptoms observed in the absence oftreatment with the particular composition or method. As used herein, theterm “reducing” symptoms refers to decreasing the levels of one or moresymptoms. The term “delaying” symptoms refers to increasing the timeperiod between exposure to the immunogen and the onset of one or moresymptoms.

The term “eliminating” symptoms refers to completely “reducing” and/orcompletely “delaying” one or more symptoms.

The term “animal” refers to any animal whose antibodies, or T cellreceptors, are capable of specifically recognizing an antigen.Alternatively, the term “animal” includes any animal which is capable ofinducing a specific humoral or cell-mediated immune response to animmunogen. Preferred animals include, but are not limited to mammalssuch as rodents, humans, primates, ovines, bovines, ruminants,lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.Preferred animals are selected from the “order Rodentia” which refers torodents that is, placental mammals (class Euthria) which include thefamily Muridae (for example, rats and mice), most preferably mice. Theterms “nucleic acid sequence” and “nucleotide sequence” as used hereinrefer to an oligonucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.

The terms “amino acid sequence,” “peptide,” “peptide sequence,”“polypeptide,” and “polypeptide sequence” are used interchangeablyherein to refer to a sequence of amino acids.

The terms “peptide of interest,” “nucleotide sequence of interest,” and“molecule of interest” refer to any peptide sequence, nucleotidesequence, and molecule, respectively, the manipulation of which may bedeemed desirable for any reason, by one of ordinary skill in the art.Exemplary molecules of interest include, but are not limited to, apeptide, glycopeptide, polysaccharide, lipopeptide, glycolipid, lipid,steroid, nucleic acid, etc.

The term “derived” when in reference to a peptide derived from a cancercell as used herein is intended to refer to a peptide which has beenobtained (for example, isolated, purified, etc.) from a cancer cell.

The term “biologically active” when applied to any molecule (forexample, polypeptide, nucleotide sequence, etc.) refers to a moleculehaving structural, regulatory and/or biochemical functions of thenaturally occurring molecule.

A peptide sequence and nucleotide sequence may be “endogenous” or“heterologous” (that is, “foreign”). The term “endogenous” refers to asequence which is naturally found in the cell or virus into which it isintroduced so long as it does not contain some modification relative tothe naturally-occurring sequence. The term “heterologous” refers to asequence which is not endogenous to the cell or virus into which it isintroduced. For example, heterologous DNA includes a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA alsoincludes a nucleotide sequence which is naturally found in the cell orvirus into which it is introduced and which contains some modificationrelative to the naturally-occurring sequence. Generally, although notnecessarily, heterologous DNA encodes heterologous RNA and heterologousproteins that are not normally produced by the cell or virus into whichit is introduced. Examples of heterologous DNA include reporter genes,transcriptional and translational regulatory sequences, DNA sequenceswhich encode selectable marker proteins (for example, proteins whichconfer drug resistance), etc.

The term “wild-type” when made in reference to a peptide sequence andnucleotide sequence refers to a peptide sequence and nucleotidesequence, respectively, which has the characteristics of that peptidesequence and nucleotide sequence when isolated from a naturallyoccurring source. A wild-type peptide sequence and nucleotide sequenceis that which is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the peptidesequence and nucleotide sequence, respectively. In contrast, the term“modified” or “mutant” refers to a peptide sequence and nucleotidesequence which displays modifications in sequence and/or functionalproperties (that is, altered characteristics) when compared to thewild-type peptide sequence and nucleotide sequence, respectively. It isnoted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type peptide sequence and nucleotide sequence.

The term “isolated” when used in relation to a molecule (for example, anucleic acid sequence, amino acid sequence, etc.) refers to a moleculethat is identified and separated from at least one contaminant moleculewith which it is associated.

As used herein, the term “purified” refers to a molecule (for example, anucleic acid sequence, amino acid sequence, etc.) that is removed fromits natural environment, isolated, or separated. An “isolated” moleculeis therefore a purified molecule. “Substantially purified” molecules areat least 60% free, preferably at least 75% free, and more preferably atleast 90% free from other components with which they are associated.

The terms “pathogen” and “animal pathogen” refer to any organism whichcauses a disease in an animal. Pathogens include, but are not limitedto, viruses, bacteria, protozoa, nematodes, fungus, etc.

The term “cancer cell” refers to a cell undergoing early, intermediateor advanced stages of multi-step neoplastic progression. The features ofearly, intermediate and advanced stages of neoplastic progression havebeen described using microscopy. Cancer cells at each of the threestages of neoplastic progression generally have abnormal karyotypes,including translocations, inversion, deletions, isochromosomes,monosomies, and extra chromosomes. Cancer cells include “hyperplasticcells,” that is, cells in the early stages of malignant progression,“dysplastic cells,” that is, cells in the intermediate stages ofneoplastic progression, and “neoplastic cells,” that is, cells in theadvanced stages of neoplastic progression.

The term “modified plant virus” refers to a plant virus, any part ofwhich has been modified by chemical, biochemical, and/or molecularbiological techniques. A “chimeric plant virus” is a plant virus whichhas been modified by means of molecular biological techniques.

The terms “TH1-type response,” and “TH1 response” when made in referenceto a response in an animal refer to any cellular and/or humoral responsewhich is generated by TH1 lymphocytes upon stimulation by an antigen,including, but not limited to, changes in the level of TH1-associatedimmunoglobulin, TH1 cell proliferation, and/or TH1-associated cytokine.In contrast, “TH2-type response,” “TH2 response” when made in referenceto a response in an animal refers to any cellular and/or humoralresponse which is generated by TH2 lymphocytes upon stimulation by anantigen, including, but not limited to, changes in the level ofTH2-associated immunoglobulin, TH2 cell proliferation, and/orTH2-associated cytokine.

The terms “TH1-associated immunoglobulin” and “TH1 cell-derivedimmunoglobulin” refer to one or more of the immunoglobulins (forexample, IgG) which are generated by TH1 cells. In contrast, the terms“TH2-associated immunoglobulin” and “TH2 cell-derived immunoglobulin”refer to one or more of the immunoglobulins (for example, IgG) which aregenerated by TH2 cells. The subtypes of TH1-associated immunoglobulinsand of TH2-associated immunoglobulins are species-specific in that theyvary from species to species. For example, whereas in mouse IgG2 (and inparticular, IgG2a and IgG2b) is the principle TH1-associatedimmunoglobulin, in man IgG1 and IgG3 are the TH1-associatedimmunoglobulins that perform TH1 functions. With respect toTH2-associated immunoglobulins, these include mouse IgG1 and IgG3, andhuman IgG2. Methods for determining immunoglobulin subtypes are known inthe art and also described herein using, for example, enzyme-linkedimmunosorbent assay (ELISA) techniques which employ coating sample wellswith commercially available alkaline phosphatase (AP)-labeled anti-IgGconjugates (for example, alkaline phosphatase (AP)-conjugated goatanti-mouse IgG₁, IgG_(2a), IgG_(2b) or IgG₃ [Southern BiotechnologiesInc., USA]) for detection using p-nitrophenyl phosphate (PNPP, Sigma) asthe substrate.

The terms “TH1-associated cytokine” and “TH1 cell-derived cytokine”refer to one or more of the cytokines produced by TH1 type cells,including, without limitation, interleukin-2 (IL-2), tumor necrosisfactor-β (TNF-β), and interferon-γ (IFN-γ). In a preferred embodimentthe TH1-associated cytokine is FN-γ. In contrast, the terms“TH2-associated cytokine” and “TH2 cell-derived cytokine” refer to oneor more of the cytokines produced by TH2 type cells, including, withoutlimitation, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6(IL-6), interleukin-9 (IL-9), interleukin-10 (IL-10), and interleukin-13(IL-13). In a preferred embodiment the TH2-associated cytokine is IL-4.

DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions which areeffective in modulating the nature and/or level of an immune response toa molecule exemplified by, but not limited to, an antigen or immunogen.In particular, the invention provides methods and means for effecting aTH1 bias in the immune response to molecules such as antigens orimmunogens, and/or reducing a TH2 bias in the immune response to suchmolecules. More particularly, the invention provides methods and meansfor increasing a TH1 immune response which is directed against moleculesthat otherwise generally stimulate a TH2-type response. The inventionfurther provides compositions and methods to reduce a TH2 immuneresponse to molecules. Additionally provided herein are compositions andmethods for altering (that is, increasing or decreasing) the level ofTH1- and TH2-associated immunoglobulins, the level of proliferation ofTH1- and TH2-associated cytokines, and the level of proliferation of TH1and TH2 cells.

In a first aspect, the invention provides modified plant virus particleswhich are capable of presenting to cells of the immune system moleculeswhich are effective as either immunogens or antigens for the generationof antibodies and/or cytokines. More particularly, molecules presentedvia the invention's modified plant virus particles stimulate a TH1-typeresponse, more preferably to stimulate a predominant TH1-type response.

In a second aspect, the present invention discloses means to modulatethe particular branch of T helper pathways to reduce or overcome anundesirable bias toward a TH2-type response which is inherent in certainmolecules. More preferably, such modulation results in exclusively orpredominantly a TH1-type response in an animal inoculated with themolecule.

In a third aspect, the invention discloses methods for modulating animmune reaction in the absence of extraneous immunomodulatory agents(for example, adjuvants, cytokines, etc.)

In a fourth aspect, methods for preventing, treating, or vaccinatingagainst diseases (exemplified, but not limited to, infectious diseases,allergies, and cancer) are described.

More particularly, the invention discloses the use of non-replicatingplant viruses as carriers of molecules (for example, antigens andimmunogens).

Data presented herein shows that, surprisingly, immunization withmodified plant viruses as a platform for molecule presentation resultsin plant virus-specific and molecule-specific TH1 cells. A profound biastowards a TH1-type immune response is shown herein using the exemplarycowpea mosaic virus (CPMV) to present peptides which are derived from awide range of sources including bacteria, virus, immunoglobulin,hormone, and cancer-associated cell surface protein. The bias in favorof a TH1-type immune response is demonstrated herein to be independentof the source of the molecule, the dosage of the molecule, the presenceor absence of adjuvant, the nature of the immunomodulating activity ofthe adjuvant if present, the route of administration, and the geneticconstitution (genotype) of the immunized animal. This result issurprising in view of the prior art's reports that the priming of THcell subsets is affected by the strain of animal used, the identity ofthe antigen, the route of antigen delivery, and the immunizationregimen. This result is also surprising in view of the non-replicationnature of the invention's modified plant viruses and the prior art'sreports that live viruses are required for a predominant TH1 response(Nguyen et al. (1994) supra).

A number of peptides are immunogenic when expressed on CPMV (McLain etal. (1995) AIDS Res Hum Retro 11:327; Dalsgaard et al. (1997) Nat.Biotechnol. 15:248; Brennan et al. (1999) Microbiol. 145:211; Brennan etal. (1999) J. Virol 73:930). Peptides derived from fibronectin-bindingprotein (FnBP) found in the outer membrane of Staphylococcus aureus andexpressed on the surface of cowpea mosaic virus elicit predominantlypeptide-specific IgG_(2a) in C57BL/6 mice (Brennan et al. (1999)Microbiology 145:211), suggesting a TH1-bias in the responses to theseparticular chimeric virus particle (CVP)-expressed peptides inoculatedinto one strain of mice. However, no cellular (T cell) responsesassociated with this particular phenomenon are reported.

In particular, the invention discloses that, surprisingly, peptidesderived from a wide range of sources including bacteria, virus,immunoglobulin, hormone and cancer-associated cell surface protein, tendto generate much higher levels of peptide-specific IgG_(2a) relative tolevels of peptide-specific IgG₁ when displayed on the exemplary cowpeamosaic virus (CPMV). This plant virus does not replicate in human cellsand thus behaves like a whole inactive virus in a mammalian system. Ofthe different CVPs tested herein, six generated a predominance ofpeptide-specific IgG_(2a) antibody over IgG_(2b) antibody, while onlyCPMV-MAST1 seemed to favor IgG_(2b) production over that of IgG_(2a)under certain conditions. The CPMV-specific antibody responses alsoshowed the same predominance of IgG_(2a) over IgG₁. At the cellularlevel, much higher numbers of both peptide- and CPMV-specificIgG_(2a)-producing spot forming cells (SFCs) [B cells] were producedcompared to IgG₁-producing SFCs in the spleens of CVP-immunized mice.

While an understanding of mechanism is not required, and withoutintending to limit the invention to any particular mechanism, the strongpolarization of the isotype response to both the peptide and virus infavor of a TH1-type response appears to be related to the ability of thevirus to prime virus-specific CD4+ TH1, thereby facilitating theclass-switching of peptide- (and virus)-specific naive B cells toTH1-associated immunoglobulin-producing plasma cells.

The predominance of peptide-specific IgG_(2a) over IgG₁ is demonstratedherein in five different inbred mouse strains encompassing the H-2^(b)(C57BL/6), H-2^(d) (BALB/c), H-2^(q) (NIH), 2^(dql) (Biozzi AB/H), andH-2^(s) (DBA/1). Even in the TH2-biased BALB/c and Biozzi AB/H mice[Natsuume-Sakai et al. (1977) Immunology 32:861; Sant'Anna et al. (1985)Immunogenetics 22:131], IgG_(2a) surprisingly predominated over IgG₁.Adjuvants tested herein with the CVPs included those which favor TH1responses such as Freund's Complete Adjuvant, and those which favor TH2responses [alum and QS-21: Cooper (1994) “The Selective Induction ofDifferent Immune Responses by Vaccine Adjuvants,” in Vaccine Design (G.I. Ada., ed.), R. G. Landes Company, pp. 125]. Again, levels of IgG_(2a)generated using these adjuvants or in the absence of adjuvant weresurprisingly much higher than levels of IgG₁, highlighting that it isindeed the carrier plant virus rather than the adjuvant that is drivingthe TH1 responses. Importantly, unlike the case with the animal virusesdescribed above, the predominance of IgG_(2a) over IgG₁ was observedirrespective of the mouse strain or the adjuvant used for immunization.

The invention further discloses that immunization of both high and lowdoses (ranging from 2-300 μg) of CVPs led to the generation of TH1-typeresponses. Thus, the dose over three orders of magnitude of administeredantigen, previously shown to influence the generation of mouse IgGisotypes (Hocart et al (1989) J. Gen. Virol. 70:2439; Hocart et al.(1989) J. Gen. Virol. 70:809), surprisingly did not appear to affect theTH1 bias in the isotypes of peptide-specific IgG.

The results obtained with the exemplary icosahedral plant virusesdescribed herein were surprising because, in part, other icosahedralvirus-like particles (VLPs) derived from animal viruses such as Norwalkvirus (Ball et al. (1998) J. Virol 72:1345) and rotavirus (O'Neal et al.(1997) J. Virol. 71:8707) do not elicit such a strong predominance ofIgG_(2a) over IgG₁ as that reported here. It is reported elsewhere thattargeting of particulate antigens to macrophages is not sufficient initself to stimulate a polarized TH1 response (Sedlik et al. (1997) Intl.Immunol. 9:91) since co-immunization with immune-modulators such asIL-12 and poly(1):(C) is required.

The compositions and methods of the invention are useful in generatingantibodies to a molecule, in inducing a desirable TH1-type responseand/or reducing an undesirable TH2-type response to a molecule for thepurpose of, for example, detecting, preventing, and/or treatingdiseases. In particular, the modified plant viruses of the inventionfind particular (although not exclusive) use in clinical applicationssince their immunomodulatory effects may be achieved in the absence ofextraneous immunomodulatory agents (such as cytokines and adjuvants),thereby avoiding the expense and adverse side effects which areassociated with administration of these agents. Moreover, because themodified plant viruses of the invention do not replicate in human cells,they represent an advantage over existing vaccine carriers since theyavoid safety concerns which are implicated in the existing live animalviruses/bacteria and DNA carrier systems.

The invention is further described under (A) Development of TH1/TH2Cells, (B) TH1-Type and TH2-Type Responses and Pathology, (C) PlantViruses, (D) Molecules of Interest, (E) Expression of Polypeptides byPlant Viruses, (F) Conjugating Molecules to Plant Viruses, (G)Administering Compositions to Animals, (H) Increasing a TH1-TypeResponse, and (D) Reducing a TH2-Type Response.

A. Development Of TH1/TH2 Cells

In mice, naive CD4+ T cells are classified into two groups, TH1 and TH2,which are characterized by differential cytokine secretion profiles andhence distinct effector functions (for review see Mosmann et al. (1986)J. Immunol. 136:2248-2357). While an understanding of mechanism is notnecessary to practice the invention, and without limiting the inventionto any particular mechanism, T cell clones which demonstrate thearchetypal TH1 or TH2 properties may be extremes of a continuum ofdifferentiated CD4+ cells and the in vivo cytokine profile that isproduced in a “typical” TH1 or TH2 response is produced by a spectrum ofcell types.

However, for simplicity it is easier to refer to the two cell types asif they are separable entities. Thus, the term “TH1 cell” as used hereinrefers to a T helper cell which produces one or more TH1-associatedimmunoglobulins (for example, mouse IgG_(2a), mouse IgG_(2b), humanIgG₁, and human IgG3) and/or one or more TH1-associated cytokines (forexample, IL-2, TNF-β, and IFN-γ). In contrast, a “TH2 cell” as usedherein refers to a T helper cell which produces one or moreTH2-associated immunoglobulins (for example, mouse IgG1, mouse IgG3, andhuman IgG2) and/or one or more TH2-associated cytokines (for example,IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13).

TH1 and TH2 cells develop from a common pool of naive CD4+ cells.Several factors may influence TH cell differentiation into the polarizedTH1 or TH2 pathway. The cytokine profile of “natural immunity” evoked bydifferent agents, the nature of the peptide ligand, the identity andlevel of microenvironmentally secreted hormones, the levels of antigen,and the presence on the T cells of co-stimulatory signaling receptorsand MHC genotype can determine the development of the subsets, with TH2cells being more dependant on high levels of antigen and the presence ofco-stimulatory molecules. For example, the prior art has observed that,in mouse vaccination models, several factors influence which TH cellsubsets are primed and the generation of specific mouse IgG isotypes.Such factors include the genetic background of the mouse strain used,the route of antigen delivery and the immunization regimen (includingantigen quantity and half life), and the nature of the antigen. TH2-typecytokine profiles often appear later than TH1 responses in vivo.

The key cytokine for TH1 generation is believed to be IL-12, produced byactivated macrophages and dendritic cells. The key cytokine for TH2generation is believed to be IL-4. IL-4 is produced in small amountsduring initial T cell activation (a particular subset of CD4 cellscalled the NK1.1 cell has been suggested as the initial source of IL-4production). The development of a TH2 response is believed to be drivenby development of local concentrations of IL-4, possibly as a result ofpersistent T cell stimulation.

With respect to the function of TH1 cells, these cells produceinterleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-β(TNF-β) which mediate macrophage and cytotoxic T cell activation (CTL)and are the principle effectors of cell-mediated immunity againstintracellular microbes and of DTH (delayed-typer hypersensitivity)reaction [Mosmann et al. (1986), supra]. CTL reactions are increasinglyrecognized as important therapeutic factors in the treatment of, orresponse to, solid tumors. IFN-γ, produced by TH1 lymphocytes, alsoinduces B cell isotype class-switching to the IgG_(2a) subclass, whichis the principal effector isotype of mouse IgG. IgG_(2a) (and to alesser extent IgG_(2b)) enhances antibody-dependent cell-mediatedcytotoxicity (ADCC; Huesser et al. (1977) J. Exp. Med. 146:1316) andstrongly binds Clq of the classical complement pathway which opsonizescells or antibody clusters for phagocytosis [Klaus et al. (1979)Immunology 38:687]. In man, IgG1 and IgG3 mediate these functions. Thusas a result of these effector functions, IgG_(2a) better protects miceagainst virus infections, tumors [Kaminski et al. (1986) J. Immunol.136:1123] and parasites [Wechsler et al. (1986) J. Immunol. 137:2968]and enhances bacterial clearance [Zigterman et al. (1989) Infect. Immun.57:2712].

In contrast to TH1 cells, TH2 cells produce interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin-13 (IL-13)which suppress cell-mediated immunity [Mosmann et al. (1986), supra].IL-4 induces B cells to produce IgG₁, which in mice poorly fixescomplement and does not mediate ADCC [Klaus et al. (1979) Immunology38:687]. It also induces the production of immunoglobulin E (IgE), whichbinds to mast cells and basophils. Consequently, TH2 cells are mainlyresponsible for phagocyte independent host defense against, for example,helminthic parasites [Sher et al. (1992) Ann. Rev. Immunol. 10:385] andin the development of allergic reactions [Erb et al. (1996) Immunol.Cell Biol. 74:206].

B. TH1-Type And TH2-Type Responses and Pathology

A number of factors (for example, type of antigen, genetic background ofthe host animal, route of administration, choice of adjuvant) arereported by the prior art to dictate the nature and extent of an immuneresponse elicited by the exposure of a molecule to an animal's immunesystem. For example, murine antibody responses to soluble proteins andto carbohydrates are generally restricted to the IgG₁ and IgG3subclasses, respectively. This suggests that IgG isotypes are notrandomly selected. Indeed, live viruses (including a range of RNA andDNA viruses) preferentially induce the production of specific IgG_(2a)antibodies [Coutelier et al. (1987) J. Exp. Med. 165:64). It is arguedthat the infection process generated by live viruses may be crucial forthe production of IFN-γ that preferentially elicits virus-specific TH1responses and the predominance of virus-specific IgG_(2a), [Nguyen etal. (1994) J. Immunol. 152:478]. This may also explain why replicating(live) bacterial and DNA vaccines generate TH1-biased responses [Londonoet al. (1996) Vaccine 14:545; Pertmer et al. (1996) J. Virol. 70:6119].However, it is known that some live viruses such as influenza virus[Balkovic et al. (1987) Antiviral Res. 8:151], herpes simplex virus typeI [HSV-1; McKendall et al. (1988) J. Gen Virol 69:847], Theiler's murineencephalomyelitis virus [Peterson et al. (1992) Immunology 75:652] andfoot-and-mouth disease virus [Perez-Filgueira et al. (1995) Vaccine13:953] elicit predominant isotypes other than IgG_(2a). In addition tothe nature of antigen, the genetic background of the host animalinfluences the nature and extent of an immune response. Thus, in thecase of influenza virus, BALB/c mice produce predominantly IgG_(2a) yetC57Ba1/6 mice produce predominantly IgG1 [Hocart et al. (1989) J. Gen.Virol. 70:2439]. Thus, the immune reaction against these live viruses issensitive to a genetic element (immunogenomic factor).

The nature of the adjuvant also plays a role in determining the natureof the immune response. For foot-and-mouth disease virus (FMDV),IgG_(2b) is the predominant isotype produced to both live and inactivewhole virus, except when a water-in-oil adjuvant is used with theinactive virus. With the latter adjuvant combination, a predominance ofIgG_(2a) (Perez-Filgueira et al. (1995) Vaccine 13:953) results. Hence,the choice of adjuvant can clearly influence the branch of the T helperresponse.

Furthermore, some inactivated viruses can elicit predominantly IgG_(2a)antibodies. So overall, a variety of studies show that the ability toelicit virus-specific IgG_(2a) may not simply be due to the infectionprocess, but may also be dependent on the nature of the virus itself,the H-2 haplotype of the mouse, the route of immunization and also onthe choice of adjuvant.

The cytokine profiles generated by the TH1- and TH2-type T cells lead todiffering physiological responses to pathogens that seek to reduce,ameliorate or remove the pathogen burden. However tissue damage can alsooccur due to persistent or over-reaction to the pathogen.

In particular, in the case of TH1, the recruitment and activation ofmacrophages can lead to undesirable granulomatous inflammation, thepre-eminent example being the tuberculoid form of leprosy. If theinfecting microorganism is an intracellular organism like the ones whichcause tuberculosis, brucellosis, or the organisms Pneumocystis carini(protozoan), a fungus like Candida albicans or Leishmania major (anintracellular parasite-protozoan), the TH1 response leads to adelayed-type hypersensitivity (DTH) response that results in theelimination of the cells containing these organisms. The release ofIFN-γ in the vicinity of the infection activates macrophages. Thelocalized release of lysosomal enzymes from the macrophage killsinfected cells and healthy bystander cells resulting in the destructionof the invading microorganism. In addition, there is a release by theTH1 cell of a protein called MIF (macrophage inhibition factor). Thisprotein is an anti-chemotactic factor which renders immobile anymacrophage in the TH1 cell's vicinity. Thus, macrophages remain at thesite of the infection. The lung damage seen due to tuberculosis andperhaps to Pneumocystis carinii is the result of indiscriminate celldamage caused by active macrophages and lysosomal enzyme release intothe tissue. TH1-dominated responses may also be involved in thepathogenesis of organ-specific autoimmune disorders, acute allograftrejection, unexplained recurrent abortions, contact dermatitis, and somechronic inflammatory disorders of unknown etiology [summarized byRomagnani (1996) Clin. Immunol. Immunopathol. 80:225].

Inappropriate TH2 responses also can result in undesirable resultsincluding recruitment of basophils and eosinophils that can have adverseconsequences in the development of allergies and asthma. The response toan early form of the RSM (respiratory syncytial virus) vaccine(containing alum-conjugated killed virus vaccine) is an example of aninappropriate TH2 response that led to cases of eosinophilia andbronchospasm. Similar reactions can lead to asthmatic symptoms. TH2-typeresponses are also responsible for Omenn's syndrome, reduced protectionagainst some intracellular pathogens, transplantation tolerance, chronicGVHD (graft versus host disease), atopic disorders, and some systemicautoimmune diseases.

It has been noted that altering the sequence (and hence affinity) ofpeptides, within the MHC class II complex can affect the nature of theCD4+ T cell response (Murray (1998) Immunol. Today 19:157). Thus peptideepitopes derived from proteins from infectious agents can becomemodified through evolution to cause the re-direction of the hostresponse to the pathogen, for example, between TH1 and TH2 responses.Parallel evolution of host and pathogen can result in the development ofT-epitopes on pathogens that bind with particular affinities to MHCsubsets. The host response must be balanced to meet the requirements ofdetecting multiple pathogens and forms of pathogens. Therefore a certaindegree of sequence variation in immunogenic determinants of a pathogenmight be expected to occur. To some extent, this represents one means bywhich the immunomodulation of a reaction to a particular immunogen mightbe contemplated that is the mutation of amino acids within a peptide toachieve a distinct type of immune reaction over that raised against thewild-type peptide.

C. Plant Viruses

The invention provides non-replicating modified plant virus particleswhich are capable of presenting to cells of the immune system moleculeswhich are effective as either immunogens or antigens to generateantibodies and/or cytokines, stimulate a TH1-type response, preferably apredominant TH1-type response, reduce an undesirable bias toward anextant, or an expected, TH2-type response which is inherent in certainmolecules.

The invention contemplates the use of any virus in which the nucleicacid coding for the capsid is a separate moiety from that which codesfor other functional molecules, and whose coat proteins have a β-barrelstructure. An advantage of the use of viruses which have this structureis that the loops between the individual strands of β-sheet provideconvenient sites for the insertion of foreign peptides. Modification ofone or more loops is a preferred strategy for the expression of foreignpeptides in accordance with the present invention. In one embodiment,the invention contemplates the use of comoviruses (such as cowpea mosaicvirus and bean pod mottle virus), nepoviruses (such as tomato ringspotvirus and strawberry latent ringspot virus), tombusviruses (such astomato bushy stunt virus (TBSV)), and sobemoviruses (such as southernbean mosaic virus (SBMV)). In particular, the tombusviruses andsobemoviruses have similar 3-dimensional structures to those ofcomoviruses and nepoviruses, but have a single type of β-barrel.

In a more preferred embodiment, the virus is a comovirus. An advantageof the comoviruses is that their capsid contains sixty copies each ofthree different β-barrels which can be individually manipulated, thusallowing expression of 60-180 copies of a peptide by a single virusparticle.

Comoviruses are a group of at least fourteen plant viruses whichpredominantly infect legumes. Their genomes consist of two molecules ofsingle-stranded, positive-sense RNA of different sizes which areseparately encapsidated in isometric particles of approximately 28 nmdiameter. The two types of nucleoprotein particles are termed middle (M)and bottom (B) component as a consequence of their behavior in caesiumchloride density gradients, the RNAs within the particles being known asM and B RNA, respectively. Both types of particle have an identicalprotein composition, consisting of 60 copies each of a large (VP37) anda small (VP23) coat protein. In addition to the nucleoprotein particles,comovirus preparations contain a variable amount of empty (protein-only)capsids which are known as top (T) component. In a preferred embodiment,the comovirus is cowpea mosaic virus (CPMV).

In the case of the exemplary member of the comovirus group, cowpeamosaic virus (CPMV), it is known that both M and B RNA arepolyadenylated and have a small protein (VPg) covalently linked to their5′ terminus. More limited studies on other comoviruses suggest thatthese features are shared by the RNAs of all members of the group. BothRNAs from CPMV have been sequenced and shown to consist of 3481 (M) and5889 (B) nucleotides, excluding the poly (A) tails. Both RNAs contain asingle, long open reading frame, expression of the viral gene productsoccurring through the synthesis and subsequent cleavage of largeprecursor polypeptides. Though both RNAs are required for infection ofwhole plants, the larger B RNA is capable of independent replication inprotoplasts, though no virus particles are produced in this case. Thisobservation, coupled with earlier genetic studies, established that thecoat proteins are encoded by M RNA.

A 3.5 A electron density map of CPMV shows that there is a clearrelationship between CPMV and the T-3 plant viruses such as thetombusviruses, in particular tomato bushy stunt (TBSV) and thesobemoviruses, in particular southern bean mosaic (SBMV). The capsids ofthese latter viruses are composed of 180 identical coat proteinsubunits, each consisting of a single β-barrel domain. These can occupythree different positions, A, B and C, within the virions. The two coatproteins of CPMV were shown to consist of three distinct P-barreldomains, two being derived from VP37 and one from VP23. Thus, in commonwith the T-3 viruses, each CPMV particle is made up of 180 β-barrelstructures. The single domain from VP23 occupies a position analogous tothat of the A type subunits of TBSV and SBMV, whereas, the N- andC-terminal domains of VP37 occupy the positions of the C and B typesubunits, respectively (U.S. Pat. No. 5,874,087; incorporated in itsentirety by reference).

X-ray diffraction analysis of crystals of CPMV and another member of thegroup, bean pod mottle virus (BPMV) shows that the 3-D structures ofBPMV and CPMV are very similar and are typical of the comovirus group ingeneral.

In the structures of CPMV and BPMV, each β-barrel consists principallyof 8 strands of antiparallel β-sheet connected by loops of varyinglength. The flat β-sheets are named the B, C, D, E, F, G, H and Isheets, and the connecting loops are referred to as the βB-βC, βB-βE,βF-βG and βH-βI loops.

The comoviruses are also structurally related to the animalpicornaviruses. The capsids of picornaviruses consist of 60 copies ofeach of three different coat proteins VP1, VP2 and VP3 each oneconsisting of a single β-barrel domain. As in the case of comoviruses,these coat proteins are released by cleavage of a precursor polyproteinand are synthesized in the order VP2-VP3-VP1. Comparison of the3-dimensional structure of CPMV with that of picornaviruses has shownthat the N- and C-terminal domains of VP37 are equivalent to VP2 andVP3, respectively, and that VP23 are equivalent to VP1. The equivalencebetween structural position and gene order suggests that VP37corresponds to an uncleaved form of the two picornavirus capsidproteins, VP2 and VP3.

One of the principal differences between the comoviruses andpicornaviruses is that the protein subunits of comoviruses lack thelarge insertions between the strands of the β-barrels found inpicornaviruses though the fundamental architecture of the particles isvery similar. The four loops (βB-βC, βD-βE, βF-βG and (βH-βI) betweenthe β-sheets are not critical for maintaining the structural integrityof the virions but, in accordance with this invention, are used as sitesof expression of heterologous peptide sequences, such as exemplaryantigenic sites which are derived from a wide range of sources includinga bacterium, a virus, an immunoglobulin, a hormone, and acancer-associated cell surface protein.

D. Molecules of Interest

The invention's modified plant virus particles are capable of presentingto cells of the immune system any molecule for the purpose of, forexample, generating antibodies and/or cytokines, increasing a TH1-typeresponse, and/or reducing a TH2-type response to the molecule. Theantibodies which are generated in response to the invention's modifiedplant virus particles may be used to isolate and purify the molecule.Alternatively, these antibodies may be used to prevent, diagnose, ortreat diseases which are associated with exposure of an animal to themolecule.

Molecules which are suitable for application in the instant inventioninclude any molecule which is capable of being presented at an exposedportion of the coat protein of the invention's viruses. Exemplarymolecules include, but are not limited to, those which contain a peptidesequence, nucleic acid sequence, polysaccharide, and/or lipid, such asglycopeptide, lipopeptide, glycolipid, etc.

Molecules which may be used to advantage in the instant inventioninclude those which are purified but whose structure is unknown, as wellas purified molecules of known structure (for example, peptides withknown amino acid sequence, nucleic acid sequences with known nucleicacid sequences, polysaccharides of known composition and structure,etc.). In a preferred embodiment, the molecules are purified and ofknown structure.

Where the molecule is a peptide, it may be presented by the invention'smodified viruses using molecular biological techniques to insert anucleic acid sequence which encodes the peptide into the virus genomesuch that the peptide is expressed at an exposed portion of the coatprotein of the invention's viruses (further described below).Alternatively, where the molecule is, or contains, a peptide sequence,nucleic acid sequence, polysaccharide, and/or lipid, such a molecule maybe chemically conjugated to a reactive peptide expressed by a plantvirus of the invention, as described below.

The invention contemplates polypeptide molecules which are derived fromany source. Without intending to limit the scope thereof, the inventioncontemplates polypeptides which are derived from cancer cells andpathogenic parasites (for example, bacteria, viruses, protozoa,nematodes, fungi, etc.), and in particular antigenic and immunogenicpeptides derived from these pathogenic parasites. Also included withinthe invention's scope are polypeptides which are associated with thedevelopment of disease, polypeptides that encode cytokines, polypeptideallergens, hormones, enzymes, growth factors, anti-idiotypic antibodies,receptors, adhesion molecules, and parts of any of the foregoingpeptides or of precursors thereof.

Polypeptides which are derived from cancer cells which are contemplatedto be within the scope of this invention are exemplified by, but notlimited to, the esophageal cancer associated antigen (U.S. Pat. No.6,069,233), the mammary-specific protein (mammaglobin) which isassociated with breast cancer (U.S. Pat. No. 5,922,836), the prostatemucin antigen which is associated with prostate adenocarcinomas (U.S.Pat. No. 5,314,996), human prostate specific antigen (PSA) (U.S. Pat.Nos. 6,100,444; 5,902,725), the SF-25 antigen of colon adenocarcinoma(U.S. Pat. No. 5,212,085), urinary tumor associated antigens (U.S. Pat.No. 5,993,828), melanogenic antigen (U.S. Pat. No. 6,087,110), theMART-1 melanoma antigen (U.S. Pat. No. 5,994,523), humantumor-associated antigen (PRAT) (U.S. Pat. No. 6,020,478), TRP-2 proteintumor antigen (U.S. Pat. No. 6,083,703), the human tumor-associatedantigen (TUAN) (U.S. Pat. No. 5,922,566), and the tumor specific Tantigen which is associated with virally-induced tumors (U.S. Pat. No.6,007,806). Each of the U.S. patents herein is incorporated in itsentirety by reference.

Exemplary polypeptides which are derived from pathogenic bacteriainclude Bordetella pertussis antigens (U.S. Pat. Nos. 4,029,766;5,897,867; 5,895,655), Mycobacterium tuberculosis antigens (U.S. Pat.No. 6,110,469), porin antigens from Bacterioides which is associatedwith ulcerative colitis and inflammatory bowel disease (U.S. Pat. No.6,033,864), Helicobacter pylori antigens (U.S. Pat. No. 6,025,164),Streptococcus antigens associated with dental caries (U.S. Pat. No.6,024,958), antigens derived from Campylobacter jejuni which isassociated with diarrheal disease (U.S. Pat. No. 5,874,300), theβ-glycoprotein cell surface antigen which is correlated with multidrugresistance in mammalian species (U.S. Pat. No. 4,837,306), a pilusantigen present in adhesion-forming bacteria (U.S. Pat. No. 4,795,803),and Moraxella catarrhalis outer membrane vesicle antigens associatedwith pulmonary disease (U.S. Pat. No. 5,993,826). Each of the U.S.patents herein is incorporated in its entirety by reference.

Polypeptides which are derived from pathogenic viruses are illustratedby, but are not limited to, polypeptides which have been isolated andpurified from viruses as exemplified by the rotavirus antigen (U.S. Pat.No. 6,110,724), Human Immunodeficiency Virus Type II (HIV-II,) antigensand simian Immunodeficiency Virus (SIV) antigens (U.S. Pat. No.5,268,265), non-A, non-B hepatitis virus antigen (U.S. Pat. Nos.4,702,909; 6,103,485), delta antigen of hepatitis D virus (U.S. Pat. No.4,619,896), influenza virus antigens (U.S. Pat. No. 6,048,537). Alsoincluded are viral polypeptides whose sequences are known, including,but not limited to, those derived from picornaviruses such asfoot-and-mouth disease virus (FMDV), poliovirus, human rhinovirus (HRV),and human papillomavirus (HPV) (U.S. Pat. No. 5,874,087), hepatitis Cvirus (HCV) antigen (U.S. Pat. No. 5,712,087), hepatitis B core antigen(U.S. Pat. No. 4,839,277), Epstein Barr virus-related antigens (U.S.Pat. No. 5,679,774), hepatitis V virus C33 antigen (U.S. Pat. No.5,985,541), cytomegalovirus (CMV) antigens (U.S. Pat. No. 6,074,817),human immunodeficiency virus type 2 antigen (HIV-2) (U.S. Pat. No.6,037,165), herpes simplex virus antigens (U.S. Pat. No. 6,013,433), andHTLV-I and HTLV-II antigens (U.S. Pat. No. 5,928,861). Each of the U.S.patents herein is incorporated in its entirety by reference.

Polypeptides within the scope of the invention, which are derived frompathogenic protozoa and nematodes include, for example, the peptideantigens derived from Plasmodium vivax which causes malaria (U.S. Pat.No. 5,874,527), Leishmania antigens which are associated withLeishmaniasis (U.S. Pat. No. 5,834,592), the antigens of the nematodeparasite Dirofilaria immitis (U.S. Pat. No. 4,839,275), antigens ofAnaplasma marginale which causes bovine anaplasmosis (U.S. Pat. No.4,956,278). Each of the U.S. patents herein is incorporated in itsentirety by reference.

Other polypeptides which are associated with the development of diseaseare also included within the scope of the invention. These include, butare not limited to, the exemplary GAGE tumor rejection antigen precursorwhich is associated with cancer development (U.S. Pat. No. 6,013,481),the antigens extracted from mammalian malpighian epithelia (for example,esophagus and epidermis) and associated with rheumatoid arthritis (U.S.Pat. No. 5,888,833), the Rh blood group antigens (U.S. Pat. No.5,840,585), antigens indicative of the presence and progression ofatherosclerotic plaque (U.S. Pat. No. 6,025,477), the IgG Fc-bindingprotein antigen associated with autoimmune diseases such as ulcerativecolitis, Crohn's disease, rheumatoid arthritis, and systemic lupus (U.S.Pat. No. 6,004,760), the Sm-D antigen associated with system lupuserythematosus (SLE) (U.S. Pat. No. 5,945,105), monocyte antigens (U.S.Pat. No. 6,124,436), the antigen associated with autoimmune inner earMeniere's disease (U.S. Pat. No. 5,885,783), the mesothelindifferentiation-associated antigen which is implicated in mesotheliomasand ovarian cancers (U.S. Pat. No. 6,083,502), the osteogenic andfibroblastic antigen (OFA) associated with bone-related diseases (U.S.Pat. No. 6,074,833), and the mast cell function-associated antigen(MAFA) which is associated with inflammatory and allergic reactions(U.S. Pat. No. 6,034,227). Each of the U.S. patents herein isincorporated in its entirety by reference.

Also included within the invention's scope are polypeptides that encodecytokines such as the exemplary interleukin-1α, interleukin-1β (U.S.Pat. Nos. 5,965,379; 5,955,476; 5,096,906), interleukin-2, interferon-α,interferon-γ, and tumor necrosis factor (U.S. Pat. No. 5,965,379),interleukin-6 (U.S. Pat. Nos. 5,965,379; 5,955,476; 5,942,220;5,460,810), the TGF-β superfamily which includes the TGF-β family (thatis, including TGFβ1, TGFβ2, TGFβ3, TGFβ4, TGFβ5, and TGFβ1.2), theinhibin family (that is, including activins and inhibins), the DPP/VG1family (that is, including bone marrow morphogenetic proteins (BMPs),DPP, and Vgl), and Mullerian Inhibiting Substance Family (that is,including Mullerian inhibiting substance (MIS)) (U.S. Pat. No.5,830,671), interleukin-11, leukemia inhibitory factor, oncostatin M,and ciliary neurotrophic factor (U.S. Pat. No. 5,460,810), andinterleukin-12 (U.S. Pat. No. 5,955,476). Each of the U.S. patentsherein is incorporated in its entirety by reference.

The invention also contemplates polypeptide allergens such as, butwithout limitation to, the vespid antigen 5 which is used to treatpatients with vespid venom allergy (U.S. Pat. No. 6,106,844), the CRXJII Cryptomeria japonica major pollen allergens (U.S. Pat. No.6,090,386), ryegrass pollen allergens Lol p lb.1 and Lol p lb.2 (U.S.Pat. No. 5,965,455), allergens of alder pollen, hazel pollen and birchpollen (U.S. Pat. No. 5,693,495), the house dust mite Dermatophagoidesfarinae Derf I and Derf II allergens, and, D. pteronssinus Der p I andDer p VII allergens (U.S. Pat. Nos. 5,958,415; 6,086,897; 6,077,518;6,077,517), cat allergen (Fel d I) (U.S. Pat. No. 5,547,669), cockroach(CR) allergens (U.S. Pat. No. 5,869,288), and peanut allergen (Ara h II)(U.S. Pat. No. 5,973,121). Each of the U.S. patents herein isincorporated in its entirety by reference.

Also included within the scope of the invention are polypeptide hormonesthat include, for example, parathyroid hormone (PTH), parathyroidhormone related peptide (PTHrp), and their synthetic analogs (U.S. Pat.Nos. 5,693,616; 6,110,892), naturally occurring human growth hormone andits variants (U.S. Pat. Nos. 5,424,199; 5,962,411), avian growth hormone(U.S. Pat. No. 5,151,511), luteinizing hormone-releasing hormone (LHRH)and its analogues (U.S. Pat. No. 5,897,863), nuclear hormone receptorprotein (U.S. Pat. No. 5,866,686), ecdysis triggering hormone (U.S. Pat.No. 5,763,400), gonadotropin releasing hormone (U.S. Pat. No.5,688,506), and melanin concentrating hormones (MCH) (U.S. Pat. No.5,049,655). Each of the U.S. patents herein is incorporated in itsentirety by reference.

Nucleic acid molecules within the scope of this invention include thosewhich encode each of the polypeptide molecules described supra.

Molecules which contain a polysaccharide and which are within the scopeof this invention are exemplified by polysaccharide and glycoproteinantigens derived from pathogenic parasites (in particular frombacteria), as well as glycoprotein hormones such as follicle stimulatinghormone (FSH), luteinizing hormone (LH), and thyroid stimulating hormone(U.S. Pat. Nos. 6,103,501; 5,856,137; 5,767,067; 5,639,640; 5,444,167;each incorporated in its entirety by reference.).

Lipid-containing molecules which find use in this invention include,without limitation, those which are derived from pathogenic parasites(for example, lipoproteins, lipopolysaccharides, etc.)

In a particularly preferred embodiment, the molecule is a peptide. In amore preferred embodiment, the peptide is derived from a bacteria (forexample, the OM protein F of Pseudomonas aeruginosa, and thefibronectin-binding protein (FnBP) of Staphylococcus aureus), a virus(for example, canine parvovirus), an immunoglobulin (for example, humanmIgE), a hormone (for example, human chorionic gonadotrophin), and acancer-associated cell surface protein (for example, epithelial growthfactor receptor).

E. Expression of Polypeptides by Plant Viruses

The plant viruses of the invention may be engineered in accordance withU.S. Pat. Nos. 5,874,087; 5,958,422 (each incorporated in its entiretyby reference) to express peptides of interest which are derived from anysource as described supra. For example, the exemplary comoviruses (forexample, CPMV) are capable of expressing externally from a single virusfrom 60 to 180 copies of a peptide (one peptide copy on each of the 60copies of the small (S) coat protein and of the 60 copies of the large(L) coat protein).

The peptides which may be incorporated into the invention's modifiedplant viruses preferably contain at least four (4) amino acids, and aresubject only to the limitation that the nature and size of the peptideand the site at which it is placed in or on the virus particle do notinterfere with the capacity of the modified virus to assemble whencultured in vitro or in vivo. While not intending to limit the inventionto any type or source of peptide, in one embodiment, the peptide is onewhose function requires a particular conformation for its activity.Biological activity of the peptide may be maintained by association ofthe peptide with a larger molecule (for example, to improve itsstability or mode of presentation in a particular biological system) aspreviously described (U.S. Pat. No. 5,958,422; incorporated in itsentirety by reference).

The plant viral nucleic acid is modified by introducing a nucleotidesequence coding for the peptide of interest either as an addition to(that is, insertion into) the existing viral genome, or as asubstitution for part of the viral genome. The choice of the method ofintroduction is determined largely by the structure of the capsidprotein and the ease with which additions or replacements can be madewithout interference with the capacity of the modified virus to assemblein plants.

In one embodiment, the nucleotide sequence coding for the peptide ofinterest is inserted into the viral genome. In a first embodiment, thesite of insertion of, or substitution with, the nucleotide sequencecoding for the peptide of interest is selected such that direct sequencerepeats flanking the site are absent. In the context of the presentinvention, the term “direct sequence repeat” when made in reference to aconstruct that contains a nucleotide sequence of interest means that anidentical oligonucleotide sequence is present on both sides of thenucleotide sequence of interest. Constructs that contain direct sequencerepeats flanking a nucleotide sequence of interest are undesirablebecause they are genetically unstable as a result of recombinationbetween the flanking sequence repeats, leading to loss of the flankednucleotide sequence, and reversion to the wild-type sequence.

In an alternative embodiment, where the foreign oligonucleotide sequenceis introduced into the plant virus genome as a substitution for part ofthe existing sequence, it is preferred that the substituted virus genomesequence does not encode an amino acid sequence in the viral coatprotein, which is important for virus replication, encapsidation, and/orpropagation in a host plant. This defect may be readily determined andavoided using methods known in the art in combination with the teachingsherein.

The nucleotide sequence encoding the peptide of interest may beintroduced into the plant virus by identifying that part of the virusgenome which encodes an exposed portion of a coat protein. The term“exposed portion of a coat protein” as used herein in reference to avirus is that part of the virus coat protein which is disposed on theouter surface of the coat protein. The location of portions of the coatprotein which are exposed, and which are therefore potentially optimumsites for introduction of the polypeptide of interest, may readily beidentified by examination of the three dimensional structure of theplant virus. In a further embodiment, the amino acid sequence of theexposed portions of a coat protein is examined for amino acids whichbreak α-helical structures because these are potentially optimum sitesfor insertion. Examples of suitable amino acids are proline andhydroxyproline, both of which whenever they occur in a polypeptide chaininterrupt the α-helix and create a rigid kink or bend in the structure.

Once a suitable site in the virus coat protein is selected in accordancewith the teachings above and those of U.S. Pat. Nos. 5,958,422 and5,874,087 (each is incorporated in its entirety by reference), thenucleotide sequence which encodes the peptide of interest may beintroduced into the viral genome at a site which encodes the desiredsite. Such introduction may be achieved by either insertion into, orsubstitution for, a viral sequence.

Where insertion is desired, this may be achieved by selecting twodifferent restriction enzyme sites and cleaving the nucleic acid usingthe selected restriction enzymes. A double stranded nucleotide sequencewhich encodes the peptide of interest is synthesized using methods knownin the art (for example, polymerase chain reaction, PCR) such thatoligonucleotides terminate in ends which are compatible with theselected restriction enzyme sites, thus allowing insertion into thecleaved virus nucleic acid. This procedure results in the introductionof a nucleotide sequence coding for the peptide of interest whileavoiding the presence of direct sequence repeats flanking the insert.Preferably, though not necessarily, complementary oligonucleotides aresynthesized in which the sequence encoding the peptide of interest areflanked by plant virus sequences so that the nucleotide sequence ofinterest is introduced as an addition to the existing nucleic acid.

In a preferred embodiment, the plant virus is CPMV and the peptide ofinterest is inserted in the βB-βC loop in the small coat protein (VP23).This loop is clearly exposed on the surface of the viral particle andcomputer modeling has shown that even large loops inserted at this siteare unlikely to interfere with the interaction between adjacent subunitsresponsible for capsid structure and stability. This loop has a uniqueNheI site at position 2708 of the M RNA-specific sequence where foreignsequences may be inserted.

Alternatively, where substitution of a viral genome sequence is desired,the viral sequence which is selected for substitution is cleaved usingappropriate restriction enzymes (for example, the sequence between theNheI and AatII restriction sites of the exemplary CPMV) and a nucleotidesequence encoding the peptide of interest is substituted therefor aspreviously described (U.S. Pat. No. 5,958,422; incorporated in itsentirety by reference).

Having determined the site and mode of introduction of the peptide ofinterest into the plant virus, manipulation of the plant virus mayproceed using previously described methods (U.S. Pat. Nos. 5,958,422 and5,874,087; each is incorporated in its entirety by reference). Forexample, where the plant virus is an RNA virus (for example, CPMV), itis necessary to express the peptide of interest using cDNA clones of theRNA. cDNA clones of CPMV RNAs M and B have been constructed, in whichthe cDNA clone of the M RNA contains an inserted oligonucleotidesequence encoding a heterologous peptide, which make use of thecauliflower mosaic virus (CaMV) 35S promoter sequence linked to the 5′ends of the viral cDNAs to generate infectious transcripts in the plant.This technique overcomes some of the problems encountered with the useof transcripts generated in vitro and is applicable to all plant RNAviruses.

Referring specifically to manipulating the genome of the exemplary CPMV,a full length cDNA clone of CPMV M RNA in the transcription vector pPM1is available (pPMM2902), as is a full length cDNA clone of CPMV BRNA(pBT7-123). A mixture of transcripts from pPMM2902 and pBT7-123 givesrise to a full virus infection when electroporated into cowpeaprotoplasts.

In order to avoid the creation of a direct repeat sequence flanking theinsert, a second restriction enzyme cutting site may be created in thenucleotide sequence of the region of the CPMV genome encoding VP23. Forexample, a single silent base change (U to C) at position 2740 of the MRNA creates a unique AatII site at amino acid valine 27 (position 2735of the nucleotide sequence). This may be achieved by site-directedmutagenesis of M13-JR-1 using methods described in U.S. Pat. No.5,874,087 (incorporated in its entirety by reference). The creation ofthe AatII site enables the nucleotide sequence encoding the six aminoacids from the native βB-βC loop in CPMV to be removed by digestion withNheI and AatII. The sequence can then be replaced by any sequence withNheI- and AatII-compatible ends.

Without intending to limit the invention to any type of plant virus, anymode of introduction of the peptide of interest into the virus, and/orany site of insertion in the virus, in a preferred embodiment, the plantvirus is cowpea mosaic virus (CPMV) and the peptide of interest isinserted between the alanine 22 (Ala²²) and proline 23 (Pro²³) residuesin the βB-βC loop of the small capsid protein (VP23) as previouslydescribed (U.S. Pat. No. 5,958,422; incorporated in its entirety byreference).

F. Conjugating Molecules to Plant Viruses

The plant viruses of the invention may be modified to present anymolecule of interest to the humoral and/or cellular components of theimmune system by chemically conjugating the molecule of interest to theplant virus as described below.

The term “conjugating” when made in reference to a molecule of interestand a virus as used herein means covalently linking the molecule ofinterest to the virus subject to the single limitation that the natureand size of the molecule of interest and the site at which it iscovalently linked to the virus particle do not interfere with thecapacity of the modified virus to assemble when cultured in vitro or invivo.

1. Reactive Peptide

In one embodiment, the invention contemplates conjugating molecules ofinterest to the virus at an exposed portion of the virus coat protein.This may be achieved by conjugating the molecule of interest to awild-type reactive peptide of the plant virus, or alternatively to aheterologous reactive peptide which is expressed on the surface of thevirus coat protein. In a preferred embodiment, the molecule of interestis conjugated to a heterologous reactive peptide that is expressed on anexposed surface of the plant coat protein.

The term “reactive peptide” refers to a peptide (whether wild-type orheterologous) which is capable of covalently binding to the molecule ofinterest. The term “capable of covalently binding” when made inreference to the interaction between a peptide and a molecule ofinterest means that the peptide covalently binds to the molecule in thepresence of suitable conditions, such as suitable concentration ofsalts, chemical reagents, temperature, pH, etc.

Reactive peptides of interest may range in size from 1 to 100, morepreferably from 1 to 50, yet more preferably from 1 to 20 amino acids.Notably, it has been established that at least 38 amino acid residuesmay be displayed at the surface of the exemplary CPMV (U.S. Pat. Nos.5,958,422 and 5,874,087; each is incorporated in its entirety byreference).

While not intending to limit the amino acids in the reactive peptide toany particular type of amino acid residue, in one preferred embodiment,the reactive peptide contains one or more “reactive amino acids,” thatis, amino acids which are capable of forming a covalent linkage with amolecule of interest either directly or indirectly, for example, via abifunctional molecule that is capable of covalent linkage with both thereactive amino acid and the molecule of interest. In a preferredembodiment, the reactive amino acid is a charged amino. A “charged aminoacid” is an amino acid which contains a net positive charge or a netnegative charge. “Positively charged amino acids,” which are alsoreferred to as “basic amino acids,” include lysine, arginine, andhistidine. “Negatively charged amino acids,” which are also referred toas “acidic amino acids,” include aspartic acid, glutamic acid, andcysteine. Given the sensitivity of the invention's plant viruses to thepresence of de-stabilizing charged residues at the capsid surface, it ispreferred, though not required, that the level of charge on the reactivepeptide is minimized. This may be achieved by including both negativelycharged and positively charged amino acids into the reactive peptide,such that the negative charge on the negatively charged amino acids isat least partially counter-balanced (that is, neutralized) by thepositive charge on the positively charged amino acids, so that thereactive peptide has a net positive or negative charge. In a morepreferred embodiment, the negative charge on the negatively chargedamino acids is completely counter-balanced by the positive charge on thepositively charged amino acids, such that the reactive polypeptidepossesses a net charge of zero.

The charged amino acids of the reactive peptide may be contiguous (thatis, two or more charged amino acids arranged in the absence ofintervening uncharged amino acid residues or uncharged amino acidanalogs), or non-contiguous (that is, two or more charged amino acidsarranged with at least one intervening uncharged amino acid residue oramino acid analog). Contiguous charged amino acids may be composed ofone (for example, Asp-Asp-Asp-Asp (SEQ ID NO:1); Arg-Arg-Arg;Lys-Lys-Lys-Lys-Lys (SEQ ID NO:2); or His-His) or more (for example,Asp-Glu; Cys-His-Lys; Lys-Arg-Arg, or Lys-Arg-His, or Lys-His-His) aminoacid residues.

Where the charged amino acids are contiguous, they may be arranged suchthat the negatively charged amino acids are contiguous with respect toone another, and the positively charged amino acids are contiguous withrespect to one another. Such a sequence is exemplified by, but notlimited to, the positively charged sequences Lys-Lys-Arg-His-Lys (SEQ IDNO:3) and Arg-Arg-His-Lys (SEQ ID NO:4), and the negatively chargedsequences Asp-Cys-Glu-Asp (SEQ ID NO:5) and Asp-Asp-Glu-Glu-Glu (SEQ IDNO:6). Alternatively, where the charged amino acids are contiguous, theymay be arranged such that the negatively charged amino acids arenon-contiguous with respect to one another, and/or the positivelycharged amino acids are non-contiguous with respect to one another.

A sequence of contiguous negatively charged amino acids may becontiguous to a sequence of contiguous positively charged amino acid asdescribed by the formula XnYn, where X is a sequence of contiguouspositively charged amino acids, Y is a sequence of contiguous negativelycharged amino acids, and n is an integer from 1 to 50, more preferablyfrom 1 to 25, yet more preferably from 1 to 10 amino acids. This isexemplified by the sequences Asp-Lys, Glu-Arg, Glu-Cys-Lys-Arg (SEQ IDNO:7), and Asp-Cys-Glu-His-Arg-Lys (SEQ ID NO:8).

Further, the sequence of contiguous charged amino acids may occur in thereactive peptide as a repeating sequence. The term “repeating sequence”when made in reference to an amino acid sequence that is contained in apeptide sequence means that the amino acid sequence is reiterated from 1to 2 times, more preferably from 1 to 10 times, and most preferably from1 to 100 times, in the peptide sequence. The repeats of the peptidesequence may be non-contiguous or contiguous. The term “non-contiguousrepeat” when made in reference to a repeating peptide sequence meansthat at least one amino acid (or amino acid analog) is placed betweenthe repeating sequences. The term “contiguous repeat” when made inreference to a repeating peptide sequence means that there are nointervening amino acids (or amino acid analogs) between the repeatingsequences.

In one preferred embodiment, the reactive peptide contains a repeatingsequence of contiguous positively charged amino acids as well as arepeating sequence of contiguous negatively charged amino acids, wherethe total number of positively charged amino acid residues in thesequence of contiguous positively charged amino acids is the same as thetotal number of negatively charged amino acid residues in the sequenceof contiguous negatively charged amino acids. In a yet more preferredembodiment, the sequence of contiguous positively charged amino acids iscontiguous with the sequence of contiguous negatively charged aminoacids. This is exemplified by the sequencesAsp-Lys-Asp-Lys-Asp-Lys-Asp-Lys-Asp-Lys-Asp-Lys (SEQ ID NO:9),Glu-Cys-Lys-Arg-Glu-Cys-Lys-Arg-Glu-Cys-Lys-Arg (SEQ ID NO:10),Asp-Cys-Glu-His-Arg-Lys-Asp-Cys-Glu-His-Arg-Lys (SEQ ID NO:11),Asp-Cys-Glu-His-Arg-Lys-Asp-Cys-Glu-His-Arg-Lys-Asp-Cys-Glu-His-Arg-Lys(SEQ ID NO:12),Cys-Asp-Asp-Glu-Cys-Lys-Arg-Arg-Arg-His-Cys-Asp-Asp-Glu-Cys-Lys-Arg-Arg-Arg-His-Cys-Asp-Asp-Glu-Cys-Lys-Arg-Arg-Arg-His (SEQ ID NO:13),Glu-Arg-Glu-Arg-Glu-Arg-Glu-Arg (SEQ ID NO:14),Asp-His-Asp-His-Asp-His-Asp-His-Asp-His (SEQ ID NO:15).

In an alternative preferred embodiment, the reactive peptide iscontemplated to contain non-contiguous negatively charged amino acidsand non-contiguous positively charged amino acids. In other words, thecharged amino acids (whether negatively or positively charged) may havedisposed between them amino acids (or amino acid analogs) that areeither uncharged (for example, glycine, alanine, valine, leucine,isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan,methionine, proline, asparagine, and glutamine) or that have a differentcharge. In a more preferred embodiment, the reactive peptide furthercontain a sequence of contiguous charged amino acids, wherein thesequence of contiguous charged amino acids consists either of contiguousnegatively charged amino acids, or of contiguous positively chargedamino acids. In a more preferred embodiment, the sequence is exemplifiedby a sequence of the general formula Asp-Glu_(n)-Gly-Lys_(n)-Asp-Glu_(n)(SEQ ID NO:16), Asp-Glu_(n)-Gly-Lys_(2n)-Asp-Glu_(n) (SEQ ID NO:17),Lys-Arg_(n)-Ser-Gly-Asp-Glu-Asp (SEQ ID NO:18),Lys-Arg_(n)-His-Pro-Met-Asp_(n)-Glu (SEQ ID NO:19), where n is aninteger of from 1 to 40. In yet a more preferred embodiment the sequenceis Asp-Glu-Gly-Lys-Gly-Lys-Gly-Lys-Gly-Lys-Asp-Glu (SEQ ID NO:20).

Any heterologous reactive peptide may be genetically engineered into aplant virus particle accordance with the teachings above and those ofU.S. Pat. Nos. 5,958,422 and 5,874,087 (each is incorporated in itsentirety by reference). In particular, the heterologous reactive peptidemay be expressed at an exposed portion of the coat protein of the plantvirus. It is preferred that the reactive peptide be inserted into theplant virus particle such that the reactive amino acids are displayed onthe coat protein of the plant virus, more preferably extending outwardsfrom the structure of the capsid, thereby facilitating access bychemical ligands and reagent to the reactive amino acids of the reactivepeptide. In one embodiment, the reactive peptide is inserted betweenalanine 22 and proline 23 of the small coat protein (VP-S) of cowpeamosaic virus.

2. Conjugating a Molecule of Interest to a Reactive Peptide

Any molecule of interest may be conjugated to a reactive peptide(whether wild-type or heterologous) which is displayed by theinvention's plant viruses using methods known in the art. For example,methods for conjugating polysaccharides to peptides are exemplified by,but not limited to coupling via alpha- or epsilon-amino groups toNaIO₄-activated oligosaccharide (Bocher et al. (1997) J. Immunol.Methods 27:191-202), using squaric acid diester(1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent (Tietze et al.(1991) Bioconjug Chem. 2:148-153), coupling via a peptide linker whereinthe polysaccharide has a reducing terminal and is free of carboxylgroups (U.S. Pat. No. 5,342,770), coupling with a synthetic peptidecarrier derived from human heat shock protein hsp65 (U.S. Pat. No.5,736,146), and using the methods of U.S. Pat. No. 4,639,512. Thesemethods may be applied to polysaccharide antigens including, forexample, the Staphylococcus epidermidis surface antigen (U.S. Pat. No.5,961,975). Each of the U.S. patents herein is incorporated in itsentirety by reference.

Methods for conjugating glycoproteins to peptides via the carbohydratemoieties of the glycoprotein have been described using, for example, thegeneration of reactive aldehydes on the carbohydrate moieties by mildoxidation with sodium periodate and subsequent reaction with peroxidasehydrazide (D'Alessandro et al. (1998) Clin. Chim. Acta 22:189-197), theuse of the hetero-bifunctional cross-linking reagent4-94-N-melaeimidophenyl)butyric acid hydrazide (MPBH) which allowscoupling of carbohydrate-derived aldehydes to free thiols (Chamow et al.(1992) J. Biol. Chem. 267:15916-15922), the organic cyanylating reagent1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to activatepolysaccharides prior to coupling to peptides under mild alkalineconditions (pH 7-9) (Lees et al. (1996) Vaccine 14:190-198), carboxylactivation or hydroxyl activation of the polysaccharide (Devi et al.(1995) Infect. Immun. 63:2906-2911), alkali treatment of thepolysaccharide prior to coupling to the peptide (Kabir (1987) J. Med.Microbiol. 23:9-18), and the methods of U.S. Pat. No. 4,639,512. Thesemethods may be applied to, for example, conjugating glycoproteinantigens (exemplified by the human immunodeficiency virus type 2 (HIV-2)antigen (U.S. Pat. No. 6,037,165), and the P-glycoprotein cell surfaceantigen (U.S. Pat. No. 4,837,306)) to the plant virus. Each of the U.S.patents herein is incorporated in its entirety by reference.

Also, either the protein or polysaccharide moiety of a glycoprotein maybe used to covalently link the glycoprotein to reactive peptides on thevirus by using prior art techniques that have been applied toconjugation of glycoproteins to glycoproteins, such as byphotoactivation to an azidobenzoyl derivative of one of theglycoproteins (Rathnam et al (1980) Biochim. Biophys. Acta 624:436-442).

Methods for conjugating proteins to proteins are described herein (thatis, conjugating a reactive heterologous peptide that contains a cysteineresidue with a protein of interest that has been activated withn-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); Example 11).Several additional methods are also known, including the method ofconjugating SL protein to protein allergens [Jahn-Schmid et al. (1996)Immunotechnology 2:103], coupling with a synthetic peptide carrierderived from human heat shock protein hsp65 (U.S. Pat. No. 5,736,146)the methods used to conjugate peptides to antibodies (U.S. Pat. Nos.5,194,254; 4,950,480), the methods used to conjugate peptides to insulinfragments (U.S. Pat. No. 5,442,043), the methods of U.S. Pat. No.4,639,512, and the method of conjugating the cyclic decapeptidepolymyxin B antibiotic to an IgG carrier using EDAC[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-mediated amide formation[Drabick et al. (1998) Antimicrob. Agents Chemother. 42:583-588]. Eachof the U.S. patents herein is incorporated in its entirety by reference.

Approaches to conjugate nucleic acids to proteins are also known in theart, such as those described in U.S. Pat. Nos. 5,574,142; 6,117,631;6,110,687; each of which is incorporated in its entirety by reference.

Methods for conjugating lipids to peptides have been described in theart including, but not limited to, the use of reductive amination and anether linkage which contains a secondary or tertiary amine (U.S. Pat.No. 6,071,532), the methods of U.S. Pat. No. 4,639,512, the methods usedfor covalently coupling peptides to unilamellar liposomes (Friede et al.(1994) Vaccine 12:791-797), of coupling human serum albumin to liposomesusing the hetero-bifunctional reagent N-succinimidyl-5-acetylthioacetate(SATA) (Kamps et al. (1996) Biochim. Biophys. Acta 1278:183-190), ofcoupling antibody Fab′ fragments to liposomes using aphospholipid-poly(ethylene glycol)-maleimide anchor (Shahinian et al.(1995) Biochim. Biophys. Acta 1239:157-167), and of coupling PlasmodiumCTL epitope to palmitic acid via cysteine-serine spacer amino acids(Verheul et al. (1995) J. Immunol. Methods 182:219-226). Each of theU.S. patents herein is incorporated in its entirety by reference.

G. Administering Compositions to Animals

In one embodiment, it is contemplated that the modified viruses of theinvention are used for the presentation of antigenic molecules as theimmunogenic component of vaccines. The invention's modified plantviruses provide an especially attractive epitope presentation system inthe context of vaccine design since they present the antigenic moleculeon the plant virus particle so that it is easily recognized by theimmune system, for example by location on an exposed part of the coatprotein of the virus. The modified viruses of the invention may beadministered to a recipient animal by any desired route (for example,intranasal, oral, parenteral, subcutaneous, intravenous, subcutaneous,intrathecal, intraperitoneal, intramuscular, etc.).

While data presented herein demonstrates that the modified plant virusesof the invention exert their effect on the cellular and/or humoralcomponents of the immune system without regard to the absence orpresence of extraneous immunomodulatory agents (for example, adjuvants,cytokines, etc.), the invention is expressly not limited to applicationof the invention in the absence of these agents. For example, becauseadjuvants function to enhance the nature of an immune response as wellas the particular pathway of the resultant immune reaction, one of skillin the art may consider inclusion of adjuvants together with themodified viruses of the invention to be desirable, regardless of theinfluence which the adjuvant has on the T helper pathway elicited.

Though the invention was illustrated using the exemplary adjuvants alum,FCA/FICA, and QS-21, it is expressly contemplated that the invention notbe limited to these adjuvants. Rather, any adjuvant of interest may beincluded, such as, but not limited to, those that contain an emulsionsystem and a synthetic resin material that is capable of complexing withantigens, hormones, drugs, and serum (U.S. Pat. No. 3,919,411),copolymers of polyoxyethylene/polyoxypropylene block copolymers (U.S.Pat. No. 6,086,899), 1H-imidazo[4,5-C-quinolin]-4-amine and itsderivatives (U.S. Pat. No. 6,083,505), mutant Escherichia coliheat-labile enterotoxin holotoxin (U.S. Pat. No. 6,033,673), formylmethionyl peptide (fMLP) (U.S. Pat. No. 6,017,537), ADP-ribosylatingexotoxin which is particularly suitable for transcutaneousadministration (U.S. Pat. No. 5,980,898), interleukin-12 (U.S. Pat. No.5,976,539), polydimethylsiloxane and a complex emulsifier (U.S. Pat. No.5,904,925), hemozoin or β-hematin (U.S. Pat. No. 5,849,307),Saccharomyces cervisiae glucan (U.S. Pat. No. 5,804,199), zinchydroxide/calcium hydroxide gel, lecithin, and polyalphaolefin (U.S.Pat. No. 5,232,690), polyoxyethylene sorbitan monoesters (PS) which areuseful for topical administration of antigens via mucosal membranes(U.S. Pat. No. 5,942,237), and transdermal liposomes (U.S. Pat. No.5,910,306). Furthermore, methods of using the crystalline bacterialsurface layers (SL) as adjuvants by conjugating antigens to SL are alsoknown in the art [Jahn-Schmid et al. (1997) International Immunology9:1867-1874]. Each of the U.S. patents herein is incorporated in itsentirety by reference.

Further, while not requiring cytokines or excipients for efficacy, themodified viruses of the invention may be co-administered using cytokineor excipient of interest. Exemplary cytokines include, withoutlimitation, interleukin-1α, interleukin-1β, interleukin-2,interleukin-11, interferon-α, interferon-γ, tumor necrosis factor, theTGF-β family, the inhibin family, the DPP/VG1 family, the MullerianInhibiting Substance Family, leukemia inhibitory factor, oncostatin M,and ciliary neurotrophic factor. Pharmaceutical excipients which mayfind use in combination with the invention's modified plant virusesinclude the illustrative microcrystalline cellulose-based excipientwhich has improved compressibility (U.S. Pat. No. 6,103,219),galactomannan hydrocolloid which is suitable for increasing the hardnessrating of a pharmaceutical tablet containing guar gum (U.S. Pat. No.6,063,402), cross-linked amylose which is useful as an excipient forslow release of active compounds from tablets or pellets (U.S. Pat. No.5,807,575), enzymatically debranched starches which are compressibleinto a tablet (U.S. Pat. No. 5,468,286), and lipid vesicle excipientswhich may be prepared in sprayable or droppable form for non-irritatingdelivery to nasal mucosa (U.S. Pat. No. 5,200,393). Each of the U.S.patents herein is incorporated in its entirety by reference.

H. Increasing a TH1-Type Response

The modified viruses of the invention find particular use in increasinga TH1 response to a molecule of interest, thus making the modifiedviruses of the invention particularly attractive carriers for moleculesthat target, for example, infectious disease, cancer, and allergy.

The term “increasing the level of TH1 response” and “increased level ofTH1 response” when made in reference to an animal's response to amodified virus containing a molecule of interest means that the level ofany one or more of the cellular and/or humoral response which isgenerated by TH1 lymphocytes upon stimulation by the molecule or thevirus is increased by any statistically significant amount as comparedto the corresponding response in a control animal. In particular, anincreased level of TH1 response in an animal that is exposed to amodified virus containing a molecule of interest refers to (a) anincreased level of TH I-associated immunoglobulin, (b) an increasedlevel of TH1-associated cytokine, and/or (c) an increased proliferationlevel of TH1 cells.

The term “increased level of TH1-associated immunoglobulin” in an animalthat is exposed to a modified virus containing a molecule of interestrefers to an increase, preferably at least a 0.1%, more preferably from0.1% to 50%, yet more preferably from 0.1% to 20%, and most preferablyfrom 0.1% to 10%, increase in the quantity of one or more of theTH1-associated immunoglobulin subclasses (for example, mouse IgG2a,mouse IgG2b, human IgG1, human IgG3, etc.), which is specific for eitherthe molecule of interest or for the virus, relative to the quantity oftotal TH1-associated immunoglobulin of the same subclass. For example,an increase of 5% in the quantity of molecule-specific (and/orvirus-specific) mouse IgG2a relative to the quantity of total mouseIgG2a in the same mouse is considered an increase in the level of TH1response in the mouse. Similarly, an increase of 1% in the quantity ofmolecule-specific (and/or virus-specific) mouse IgG2b relative to thequantity of total mouse IgG2b in the same mouse is considered anincrease in the level of TH1 response in the mouse (see, for example,Table 4).

Alternatively, the term “increased level of TH1-associatedimmunoglobulin” in an animal that is exposed to a modified viruscontaining a molecule of interest refers to an increase, preferably atleast a 2 fold, more preferably from 2 to 100,000 fold, more preferablyfrom 2 to 10,000 fold, and most preferably from 2 to 2,000 fold,increase in the ratio of the quantity of one or more of theTH1-associated immunoglobulin subclasses (for example, mouse IgG2a,mouse IgG2b, human IgG1, human IgG3, etc.) which is specific for eitherthe molecule of interest or for the virus, relative to the quantity oftotal TH1-associated immunoglobulin of the same subclass, on the onehand, as compared to the ratio of one or more of the TH2-associatedimmunoglobulin subclasses (for example, mouse IgG1, mouse IgG3, humanIgG2, etc.), which is specific for either the molecule of interest orfor the virus (respectively), relative to the quantity of totalTH2-associated immunoglobulin of the same subclass, on the other hand.For example, an increase of 1,000 fold in the ratio of molecule-specific(and/or virus-specific) mouse IgG2a:total IgG2a, relative to the ratioof molecule-specific (and/or virus-specific) mouse IgG1:total IgG1 inthe same mouse is considered an increase in the level of TH1 response inthe mouse. Similarly, an increase of 2,000 fold in the ratio ofmolecule-specific (and/or virus-specific) mouse IgG2b:total IgG2b,relative to the ratio of molecule-specific (and/or virus-specific) mouseIgG3:total IgG3 in the same mouse is considered an increase in the levelof TH1 response in the mouse (see, for example, Table 4).

In yet another alternative, the term “increased level of TH I-associatedimmunoglobulin” in an animal that is exposed to a modified viruscontaining a molecule of interest refers to an increase, preferably atleast 2 fold, more preferably from 2 to 10,000 fold, yet more preferablyfrom 2 to 1000 fold, even more preferably from 2 to 100 fold, and mostpreferably from 2 to 50 fold, increase in the geometric mean end-pointtiter of molecule-specific (and/or virus-specific) TH1-associatedimmunoglobulins as compared to the geometric mean end-point titer of themolecule-specific (and/or virus-specific) TH1-associated immunoglobulinsin a control animal. The term “end-point titer” is that dilution ofantibody which is specific for a given molecule and which is the highestdilution of the antibody that produces a detectable reaction (forexample, by ELISA) when combined with the molecule. For example, anincrease of 20 fold in the geometric mean end-point titer ofmolecule-specific mouse IgG2a in a treated mouse as compared to thegeometric mean end-point titer of the molecule-specific mouse IgG2a in acontrol mouse is considered an increased level of TH1-associatedimmunoglobulin (see, for example, Tables 2 and 3).

Methods for quantitating immunoglobulin levels produced by individual Bcells (as well as cells fused with B cells such as hybridomas) arereadily achieved in vitro using commercially available reagents and avariety of tests, including those described herein, such as ELISA andELISPOT. See Segwick et al. (1983) J. Immunol. Methods 57:301-309. Seealso Mazer et al. (1991) J. Allergy Clin. Immunol. 88:235-243.

In yet another alternative, an “increased level of TH1 response” refersto an increase in the level of TH1-associated cytokine. The term“increase in the level of TH1-associated cytokine” in an animal that isexposed to a modified virus containing a molecule of interest means thatthe amount of a TH1-associated cytokine which is produced by theanimal's TH1 cells is increased preferably by at least 2 fold, morepreferably from 2 to 10,000 fold, yet more preferably from 2 to 1,000fold, and most preferably from 1 to 100 fold, in a treated animalrelative to the amount of TH1-associated cytokine which is produced by Tcells of a control animal. The quantity of cytokines may be determinedusing, for example, ELISA, as described herein using commerciallyavailable reagents (for example, Table 6).

In a further alternative, an “increased level of TH1 response” refers toan increased proliferation level of TH1 cells. The term “increasedproliferation level of TH1 cells” in an animal that is exposed to amodified virus containing a molecule of interest means that the numberof proliferating TH1 cells which are produced by the animal is increasedpreferably by at least 2 fold, more preferably from 2 to 10,000 fold,more preferably from 2 to 1,000 fold, and most preferably from 1 to 100fold, relative to the number of proliferating TH1 cells which areproduced by a control animal. The number of proliferating TH1 cells maybe determined using methods such as those described herein, and theirTH1 type may be determined by examination of supernatants of these cellsfor the presence of TH1-associated cytokines (for example, Table 6).

In one embodiment, it is contemplated that a therapeutic amount of themodified plant viruses of the invention be administered to a subject.

Data presented herein demonstrates that immunization of mice with theexemplary chimeric virus particles (CVPs) of CPMV generates primarilyCPMV- and peptide-specific IgG_(2a) and IgG_(2b) antibodies in sera asdetermined by ELISA. Enzyme-linked immunospot (ELISPOT) analysisconfirmed the bias in the antibody responses toward the TH1-type,demonstrating that the CVPs can prime predominantly CPMV- andpeptide-specific IgG_(2a)- and IgG_(2b)-producing B cells in a spleen.In contrast, only low levels of CPMV- and peptide-specific IgG₁ and IgG₃antibody-producing B cells (products of the TH2 immune pathway) weredetected, if at all.

Furthermore, the invention discloses that spleen cells (T cells) fromCVP-immunized mice, proliferated to CPMV in vitro producing high levelsof IFN-γ (a TH1-associated cytokine) but no detectable levels of IL-4 (aTH2-associated cytokine). This suggests that the CVPs elicit a TH1-typeresponse to the viral carrier that, in turn, governs the isotype of thepeptide-specific B cell responses. The bias in the response towards theTH1-type was unaffected by the nature of the antigen, the geneticbackground of the individual inoculated, the choice of adjuvant, or thedose or the regimen of doses of CVPs administered.

While data presented herein demonstrates that, in a preferredembodiment, the level of TH1-associated cytokine (for example, IFN-γ)increased in response to treatment with the modified viruses of theinvention in the absence of a change in the level of TH2-associatedcytokine (for example, IL-4) it is expressly contemplated that theinvention is not limited to an increase in TH1-associated cytokine inthe total absence of detectable levels of TH2-associated cytokine.Rather, the invention expressly includes within its scope an increase inthe level of TH1-associated cytokine regardless of the change (if any)in the level of TH2-associated cytokine.

The above data demonstrates the ability of the invention's modifiedviruses, which do not infect or replicate in mammalian cells, to directthe immune response to expressed peptides toward the TH1 effector typewithout the need for extraneous immunomodulatory agents, such asadjuvants or cytokines, and represents an advantage of the invention'splant viruses as a vaccine carrier system.

Put another way, the modified viruses provided herein behave as inactiveviruses in the context of a mammalian inoculation. While inactiveviruses are predicted by the prior art to trigger a TH2 response, theinactive viruses provided herein surprisingly elicit a TH1-typeresponse.

I. Reducing a TH2-Type Response

The modified viruses of the invention are useful in applications whereit is desirable to reduce a TH2 response to a molecule of interest. Forexample, where administration of a molecule (for example, bacterialantigen) to an animal is known to generate a TH2 response (whetherpartial, predominant, or exclusive), the TH2 response in another animalmay be reduced by presenting the molecule to the other animal in thecontext of a modified plant virus as described herein.

The terms “partial TH1 response” and “partial TH2 response” mean thatthe animal exhibits (a) TH1- and TH2-associated immunoglobulin, (b) TH1-and TH2-associated cytokine, and/or (c) TH1 and TH2 cell proliferation.

A “predominant TH2 response” is a partial TH2 response in which thelevel of any one or more of TH2-associated immunoglobulin,TH2-associated cytokine, and TH2 cell proliferation is statisticallygreater than the level of TH1-associated immunoglobulin, TH1-associatedcytokine, and TH1 cell proliferation, respectively. In contrast, a“predominant TH1 response” is a partial TH1 response in which the levelof any one or more of TH1-associated immunoglobulin, TH1-associatedcytokine, and TH1 cell proliferation is statistically greater than thelevel of TH2-associated immunoglobulin, TH2-associated cytokine, and TH2cell proliferation, respectively.

An “exclusive TH2 response” is a predominant TH2 response where theanimal exhibits TH2-associated immunoglobulin, TH2-associated cytokine,and TH2 cell proliferation in the total absence of TH1-associatedimmunoglobulin, TH1-associated cytokine, and TH1 cell proliferation.Conversely, an “exclusive TH1 response” is a predominant TH1 responsewhere the animal exhibits TH1-associated immunoglobulin, TH1-associatedcytokine, and TH1 cell proliferation in the total absence ofTH2-associated immunoglobulin, TH2-associated cytokine, and TH2 cellproliferation.

Furthermore, the invention's modified viruses are also useful where itis desirable to reduce an extant TH2 in an animal. This is of particularuse in applications where booster vaccinations follow a primaryvaccination that employed an adjuvant which elicits an undesirablepartial, predominant, or exclusive TH2 response. The prior art has notedthat a pre-existing TH2 response cannot be overcome by boosterapplication of adjuvant which would otherwise result in a TH1 responsewhen administered in the primary vaccination. Specifically, while usingQuil-A as adjuvant resulted in induction of TH1 responses in mice whenimmunized with HPV 16 E7 protein, this effect was not seen when therewas a pre-existing TH2 response to E7 (induced using algammulin asadjuvant) [Fernando et al. (1998) Scand. J. Immunol. 47:459]. Thisobservation indicates that where the primary vaccination of humans hasbeen done with alum (which is the only adjuvant approved for human useand which induces TH2 responses), the resulting undesirable TH2 responsewhich mediates undesirable allergic reactions may heretofore not bereversible. While primed TH2 cells, unlike TH1 cells, are stable and maynot be directed toward the TH1 phenotype [Perez et al. (1995) Intl.Immunol. 7:869], it has been shown that in human T cell lines and clonesgenerated from allergic patients, the use of the specific allergenconjugated to a bacterial protein results in the expansion ofallergen-specific TH1/THO cells while the unconjugated allergen expandedTH2 cells [Jahn-Schmid et al. (1997) Intl. Immunol. 9:1867]. Thus, theimmunomodulatory dominance of the CPMV-presentation of immunogens(including, but not limited to, allergens) can be extended to shift anestablished immune response from a deleterious TH2-pathway to a morebeneficial TH1 pathway. Thus, the dominant immunomodulatory effect ofthe invention's modified plant viruses when used as the presentationplatform for an antigen offers a means to overcome the extant TH2response to that antigen which is initiated by the presence of aparticular adjuvant.

The term “reducing the level of TH2 response” and “reduced level of TH2response” when made in reference to an animal's response to a modifiedvirus containing a molecule of interest means that any one or more ofthe cellular and/or humoral response which is generated by TH2lymphocytes upon stimulation by the molecule or the virus is reduced byany statistically significant amount as compared to the correspondingresponse in a control animal. In particular, a reduced level of a TH2response in an animal that is exposed to a modified virus containing amolecule of interest refers to (a) a reduced level of TH2-associatedimmunoglobulin, (b) a reduced level of TH2-associated cytokine, and/or(c) a reduced proliferation level of TH2 cells.

The term “reduced level of TH2-associated immunoglobulin” in an animalthat is exposed to a modified virus containing a molecule of interestrefers to a reduction of, preferably from 0.1% to 100%, more preferablyfrom 0.1% to 80%, yet more preferably from 0.1% to 60%, in the quantityof one or more of the TH2-associated immunoglobulin subclasses (forexample, mouse IgG1, mouse IgG3, human IgG2, etc.), which is specificfor either the molecule of interest or for the virus, relative to thequantity of total TH2-associated immunoglobulin of the same subclass.For example, a reduction of 10% in the quantity of molecule-specific(and/or virus-specific) mouse IgG1 relative to the quantity of totalmouse IgG1 in the same mouse is considered a reduced level of TH2response in the mouse. Similarly, a reduction of 0.1% in the quantity ofmolecule-specific (and/or virus-specific) mouse IgG3 relative to thequantity of total mouse IgG3 in the same mouse is considered a reducedlevel of TH2 response in the mouse.

Alternatively, the term “reduced level of TH2-associated immunoglobulin”in an animal that is exposed to a modified virus containing a moleculeof interest refers to a reduction by, preferably at least 2 fold, morepreferably from 2 to 100,000 fold, yet more preferably from 2 to 10,000fold, and most preferably from 2 to 2,000 fold, in the ratio of one ormore of the TH2-associated immunoglobulin subclasses (for example, mouseIgG1, mouse IgG3, human IgG2, etc.), which is specific for either themolecule of interest or for the virus, relative to the quantity of totalTH2-associated immunoglobulin of the same subclass, on the one hand,relative to the ratio of the quantity of one or more of theTH1-associated immunoglobulin subclasses (for example, mouse IgG2a,mouse IgG2b, human IgG1, human IgG3, etc.) which is specific for eitherthe molecule of interest or for the virus (respectively), relative tothe quantity of total TH2-associated immunoglobulin of the samesubclass, on the other hand. For example, a reduction of 1,000 fold inthe ratio of molecule-specific (and/or virus-specific) mouse IgG1:totalIgG1, relative to the ratio of molecule-specific (and/or virus-specific)mouse IgG2a:total IgG2a in the same mouse is considered a reduced levelof TH2 response in the mouse. Similarly, a reduction of 2,000 fold inthe ratio of molecule-specific (and/or virus-specific) mouse IgG3:totalIgG3, relative to the ratio of molecule-specific (and/or virus-specific)mouse IgG2a:total IgG2a in the mouse is considered a reduced level ofTH2 response in the mouse.

In a further alternative, the term “reduced level of TH2-associatedimmunoglobulin” in an animal that is exposed to a modified viruscontaining a molecule of interest refers to a reduction by, preferablyat least 2 fold, more preferably from 2 to 10,000 fold, even morepreferably from 2 to 1,000 fold, and yet more preferably from 2 to 100fold, in the geometric mean end-point titer of molecule-specific (and/orvirus-specific) TH2-associated immunoglobulins as compared to thegeometric mean end-point titer of the molecule-specific (and/orvirus-specific) TH2-associated immunoglobulins in a control animal. Forexample, a reduction by 2 fold in the geometric mean end-point titer ofmolecule-specific mouse IgG1 in a treated mouse as compared to thegeometric mean end-point titer of the molecule-specific mouse IgG1 in acontrol mouse is considered a reduced level of TH2-associatedimmunoglobulin.

In yet another alternative, a “reduced level of TH2 response” refers toa reduced level of TH2-associated cytokine. The term “reduced level ofTH2-associated cytokine” in an animal that is exposed to a modifiedvirus containing a molecule of interest means that the amount of aTH2-associated cytokine which is produced by the animal's TH2 cells isreduced preferably by at least 2 fold, more preferably from 2 to 10,000fold, yet more preferably from 2 to 1,000 fold, and most preferably from1 to 100 fold, in a treated animal relative to the amount ofTH2-associated cytokine which is produced by T cells of a controlanimal.

In a further alternative, a “reduced level of TH2 response” refers to areduced proliferation level of TH2 cells. The term “reducedproliferation level of TH2 cells” in an animal that is exposed to amodified virus containing a molecule of interest means that the numberof proliferating TH2 cells which are produced by the animal is reducedpreferably by at least 2 fold, more preferably from 2 to 10,000 fold,more preferably from 2 to 1,000 fold, and most preferably from 1 to 100fold, in the treated animal relative to number of proliferating T cellswhich are produced by a control animal. The number of proliferating Tcells may be determined using methods such as those described herein,and their TH2 type may be determined by examination of supernatants ofthese cells for the presence of TH2-associated cytokines.

EXPERIMENTAL

The following Examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

Unless otherwise stated, the experimental procedures and materials ineach of the following Examples conform to the general descriptionslisted below.

Experimental Animals

Female C57BL/6 (H-2^(b), BALB/c (H-2^(d)), NIH(H-2^(q)), DBA/1(H-2⁵) andBiozzi AB/H(H-2^(dql)) mice, aged 6-8 weeks, were housed at theDepartment of Pathology, University of Cambridge, United Kingdom. Allprocedures were performed according to the United Kingdom Home Officeguidelines for animals in medical research.

Construction, Propagation and Purification of CVPs

The methods used for the expression of foreign peptides on both the Sand L subunits of CPMV were as described in Porta et al. (1994) Virology202:949. The various specific CVPs used in this investigation aredescribed in Table 1.

TABLE 1 CVPs used for immunization CVP Foreign sequence CPMV-PAE5 Aminoacids 282-295 (NEYGVEGGRVNAVG; SEQ ID NO:21) of the outer membraneprotein F (OM protein F) of Pseudomonas aeruginosa linked by S and Gresidues to residues 305-318 (NATAEGRAINRRVE; SEQ ID NO:22) of protein Fon L subunit of CPMV CPMV-MAST1 Amino acids 1-30 of thefibronectin-binding (FnBP) of Staphylococcus aureus;GQNNGNQSFEEDTEKDKPKYEQGGNIIDID (SEQ ID NO:23) on the S subunit of CPMVCPMV-HCG1 C-terminal 37 amino acids of the β subunit of human chorionicgonadotrophin (βhCG-CTP37); TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQID NO:24) on the S subunit of CPMV CPMV-HCG3 Amino acids 109-118 and132-145 from the C-terminus of βhCG (truncated form of the CTP37peptide): TCDDPRFQDSSRLPGPSDTPILPQ (SEQ ID NO:25) on the S subunit ofCPMV CPMV-AGY2 A 14 amino acid peptide (ELDVCVEEAEGEAP; SEQ ID NO:26)from the Cε4 extracellular segment of human mIgE on the S subunit ofCPMV CPMV-EGFR1 The first 13 amino acids (LEEKKGNYVVTDH; SEQ ID NO:27)of the N-terminus of human mutant epidermal growth factor receptorvariant III (EGFRvIII) on the S subunit of CPMV CPMV-PARVO9 Amino acids3-19 (DGAVQPDGGQPAVRNER; SEQ ID NO:28) from the 3L17 peptide derivedfrom the VP2 protein of canine parvovirus on both the S and L subunitsof CPMV

ELISA to Determine Peptide-Specific and Total Isotype Concentrations

In some cases the OD₄₀₅ values reported in the examples which followwere converted into micrograms of antibody. Where this was done thefollowing procedure was used as the basis for the calculation: wellswere coated with either goat anti-mouse IgG (Southern BiotechnologiesInc., USA) or with peptide. Serial dilutions of a known concentration ofan IgG_(2a) kappa mAB (Sigma, UK) were added to wells coated withanti-mouse-IgG and dilutions of CVP-immunized sera were added to wellscoated with either anti-mouse IgG or with peptide. The ELISA was carriedout as reported elsewhere in the specification using the alkalinephosphatase (AP)-labeled anti-mouse IgG_(2a) conjugate for detection. Byplotting the OD₄₀₅ obtained from the interaction of the goat anti-mouseIgG and monoclonal IgG_(2a) against the known concentrations ofIgG_(2a), the OD₄₀₅ units of anti-peptide IgG_(2a) in test serum wereconverted to micrograms of peptide-specific IgG_(2a). An OD₄₀₅ was readat a suitable point on the IgG_(2a) standard curve. Using the samestandard curve, OD₄₀₅ units from sera incubated in wells coated withanti-IgG, can be converted into micrograms of total IgG_(2a) present inthe serum sample. The percentage of peptide specific IgG_(2a) wascalculated for each serum sample. The same method utilized to measuretotal and peptide-specific IgG₁, IgG_(2b) and IgG₃.

Statistics

Differences between groups were evaluated using the student's t-testwhere P<0.05 was considered statistically significant.

Example 1 DT- and KLH-Conjugated Peptides do not Elicit DominantTH1-Type Serum Antibody Responses

Any bias seen in the T helper pathway of an immune response generated byan antigen may be governed by the intrinsic immunological properties ofthe peptide concerned. To test this, C57BL/6 mice were immunized withthe CTP37 peptide, derived from human chorionic gonadotrophin,conjugated to diphtheria toxin (DT; Prof. V. Stevens, Ohio StateUniversity), or with a peptide (peptide 10) derived from an outermembrane protein (Omp F protein) of Pseudomonas aeruginosa conjugated toKLH (Prof. H. E. Gilleland, Louisiana State University). Both conjugateswere inoculated in the presence of the adjuvant QS-21. Either twoimmunizations (on days 0 and 21) or three immunizations (on days 0, 14and 28) were administered subcutaneously. Blood was collected bytail-bleeding or following exsanguination on day 42 and sera werecollected and stored for later ELISA determinations at −20° C. For thedetection of antibodies to P. aeruginosa OM protein F and PhCG-CTP37(peptides were synthesized and purified by Genosys Inc., Cambridge, UK),microtiter plate wells were coated with 0.5 μg/well of the respectivepeptides (see Table 1, supra) for 3 h at 37° C. A series of doublingdilutions of serum were incubated on the antigen-coated plates for 1 hat 37° C. Bound antibody was detected with either alkaline phosphatase(AP)-conjugated goat anti-mouse IgG₁, IgG_(2a), IgG_(2b) or IgG₃(Southern Biotechnologies Inc., USA) using p-nitrophenyl phosphate(PNPP, Sigma) as the substrate. These anti-mouse isotype reagents werefirst titrated against standard mouse myeloma proteins representing thefour IgG subclasses and a dilution of 1:2000 of each conjugate givesless than 10% variation between myeloma binding. This was the dilutionused in all subsequent assays. End-point titers were calculated asdescribed previously (Brennan et al. (1999) Microbiol. 145:211; Brennanet al. (1999) J. Virol 73:930)). The results are shown in Table 2.

TABLE 2 DT- and KLH-conjugated peptides do not elicit dominant TH1-typeserum antibody responses Immunization schedule Mean peptide-specifictiter ± SD Conjugate Strain Regimen^(a) Adjuvant IgG₁ IgG_(2a) IgG_(2b)IgG₃ *DT- C57BL/6 2 × 10 μg QS-21 400661± 141655± 221702± 43372± •HCG-CTP37 KLH-OM C57BL/6 2 × 10 μg QS-21  48337±  7106±  26210±  1346±protein F ^(a)Immunization of C57BL/6 mice used six animals/group. *Serawere collected on day 29 and examined for βhCG-CTP37- and OM proteinF-specific IgG₁, IgG_(2a), IgG_(2b) or IgG₃ by ELISA.

As shown in Table 2, both DT-PhCG-CTP37 and KLH-OM protein F elicitedsignificantly higher levels of peptide-specific IgG₁ compared toIgG_(2a) (P<0.05 and P<0.01 for DT-PhCG-CTP37 and KLH-OM protein F,respectively), demonstrating that presentation of these peptides onnon-replicating vaccine carrier systems does not generate a bias towardsa TH1-type response.

Example 2 Expression of Peptides on CPMV Overcomes a TH2 Bias in theImmune Response Stimulated by the Peptides on other MacromolecularCarrier Systems Leading to a TH1-Type Response

In contrast to the previous example, four peptides, including the two(DT-βhCG-CTP37 and KLH-OM protein F) from Example 1 were expressed onCPMV. Four groups of eight BALB/C mice were immunized subcutaneously inthe presence of FIA/FCA in a total volume of 100 μl per dose. Threeimmunizations (on days 0 and 21 or on days 0, 14 and 28) were conductedinjecting respectively, 100 μg, 25 μg and a further 25 μg of CVPs. Bloodwas collected by tail-bleeding or exsanguination on day 42; sera werecollected and stored at −20° C.

For the detection of anti-CPMV antibody, wells were coated with 0.1μg/well of CPMV for 3 h at 37° C. A series of doubling dilutions ofserum were incubated on the antigen-mated plates for 1 h at 37° C. Boundantibody was detected with either alkaline phosphatase (AP)-conjugatedgoat-anti-mouse IgG₁, IgG_(2a), IgG_(2b), or IgG₃ (SouthernBiotechnologies Inc., USA) with p-nitrophenylphosphate (PNPP) (Sigma) asthe substrate. As before, these anti-mouse isotope reagents were firsttitrated against standard mouse myeloma proteins representing the fourIgG subclasses. A dilution of 1:2000 of each conjugate gave less than10% variation between myeloma binding; therefore this dilution was usedin all subsequent assays. End-point titers were calculated as describedpreviously. For CPMV-specific titers, the results were expressed asend-point titer, calculated as the inverse of the dilution that gives amean OD₄₀₅ higher than the OD₄₀₅ obtained with a 1:50 dilution of pooledserum from unimmunized mice.

For peptide-specific titers, end-point titers were the inverse of thedilution that gives a mean OD₄₀₅ higher than the OD₄₀₅ obtained with a1:50 dilution of pooled serum from wild-type CPMV-immunized mice. Theresults are demonstrated in Table 3, rows 5 and 7.

TABLE 3 Isotype of peptide-specific serum IgG elicited by CVPsexpressing a variety of different peptides Immunization schedule Meanpeptide-specific titer ± SD Group. CVP^(a) Strain^(b) Regimen^(c)Adjuvant^(d) IgG₁ IgG_(2a) IgG_(2b) IgG₃ 1. AGY2+ BALB/C 100/25/25FCA/FICA 793± 10832±  238± 131± 2. EGFR1 DBA/1 3 × 100 μg QS-21 405±10930±  446± NT 3. PARVO9 NIH 2 × 100 μg QS-21 346± 26365±  5157± 412±4. PARVO9* NIH 2 × 100 μg None 372± 14589±  2579± 313± 5. HCG1 BALB/c100/25/25 QS-21 498± 14172±  613± 205± 6. HCG3 C57BL/6 100/25/25 QS-21499± 21040± 15116± 919± 7. PAE5 C57BL/6 3 × 50 μg QS-21 151± 19751± 4276± 762± 8. MAST1 C57BL/6 2 × 5 μg None 2483±  16890± 44572± 3553± 9. MAST1 C57BL/6 2 × 5 μg Alum 1426±  10770± 20770± 357± 10. MAST1C57BL/6 2 × 5 μg QS-21 17704±  162696±  197958±  5840±  11. MAST1C57BL/6 2 × 5 μg QS-21 1080±  32650± 39727± 348± 12. MAST1 Biozzi 3 × 10μg QS-21 200    1021±  6552±  87± Mouse strains^(b) of different H-2haplotypes were immunized^(a) (5-9/group) subcutaneously or*intranasally with a number of CVPs^(a) in either FCA/FICA, QS-21 oralum adjuvants or without adjuvant^(d). Sera were collected on day 42(+day 83) and examined for mIgE-(AGY2), EGFRvIII-(EGFR1), VP2-(PARVO9),βhCG-CTP37-(HCG1), OM protein F-(PAE5) and FnBP-(MAST1)-specific-IgG₁,IgG_(2a), IgG_(2b), or IgG₃ by ELISA. Titers are expressed as geometricmean end-point titres ± SD.

Mouse strains^(b) of different H-2 haplotypes were immunized^(a)(5-9/group) subcutaneously or *intranasally with a number of CVPs^(a) ineither FCA/FICA, QS-21 or alum adjuvants or without adjuvant. Sera werecollected on day 42 (+day 83) and examined for mIgE-(AGY2),EGFRvIII-(EGFR1), VP2-(PARVO9), βhCG-CTP37-(HCG1), OM protein F-(PAE5)and FnBP-(MAST1)-specific-IgG₁, IgG_(2a), IgG_(2b), or IgG₃ by ELISA.Titers are expressed as geometric mean end-point titres ±SD.

The term “end-point titre” is that dilution of antibody which isspecific for a given antigen and which is the highest dilution of theantibody that produces a detectable reaction when combined with theantigen.

As demonstrated by Table 3, in this case where epitopes were presentedon CPMV, these epitopes elicited much lower levels of IgG₁ thanIgG_(2a), demonstrating a bias towards a TH1 type immune response.Indeed, in contrast to the two epitopes (that is, DT-PhCG-CTP37 andKLH-OM protein F) which were presented in the absence of CPMVpresentation (Example 1), presentation of these same epitopes on CPMVelicited much lower levels of IgG₁ than IgG_(2a).

Example 3 The Presentation of Peptides on CPMV Elicits a TH1-TypeResponse in the Presence of Extraneous Immunomodulatory Agents, forExample, Specific Adjuvants known to favor TH2-Type Immune Responses

The adjuvant alum generally favors the induction of a TH2-type immuneresponse. In order to determine whether the TH2-type immune responsewhich is favored by adjuvants could be bypassed by the invention's CPMVpresentation system, three groups of C57BL/6 mice were immunizedsubcutaneously on days 0 and 21 on each occasion with 5 μg CPMV-MAST1(expressing a peptide derived from the fibronectin-binding protein ofStaphylococcus aureus) either alone or with alum or QS-21. Sera werecollected on day 42 and assayed for MAST1 peptide-specificimmunoglobulins of the classes: IgG₁, IgG_(2a), IgG_(2b), or IgG₃ byELISA, essentially as described in Examples 1 and 2, above. The titersindicated a strong bias towards a TH1 response in all three groups ofmice including the control group in which no adjuvant was added (Table3, supra, rows 8, 9 and 10). Thus the immunomodulatory affect ofadjuvants known to favor TH2 responses was completely bypassed by thepresentation of peptides on chimeric CPMVs. Also, the absence of anextraneous adjuvant in part of this investigation serves to emphasizethe intrinsic TH1-biasing effect of the CPMV platform itself (Table 3,supra, row 8).

Example 4 Peptides Derived from a Variety of Proteins ElicitPredominantly Peptide-Specific TH1-Type Antibodies in Sera whenExpressed on CPMV Independently of the Genetic Background of theImmunized Individual

To verify the universal utility of CPMV as a carrier capable ofproducing a TH1-type response, and in order to demonstrate that theimmunological effect was independent of the genetic constitution of animmunized individual, mice of different MHC Class II haplotypes wereinoculated with CVPs expressing peptides derived from a number ofdifferent sources. CPMV-AGY2 (Table 3, row 1) expresses a peptide fromthe heavy chain of human membrane-bound immunoglobulin E (IgE);CPMV-EGFR1 (Table 3, row 2) expresses a peptide derived from protein,epithelial growth factor receptor, present on human cancer cells;CPMV-HCG3 (Table 3, row 6) express peptides derived from the humanhormone, chorionic gonadotrophin (hCG); CPMV-PARVO9 (Table 3, rows 3 and4) expresses a peptide from canine parvovirus, and CPMV-MAST1 (Table 3,rows 8-12) and CPMV-PAE5 (Table 3, row 7) express peptides derived frombacterial membrane proteins (of S. aureus and P. aeruginosa,respectively). Four mouse strains representative of different haplotypes(genotypes) were used: DBA/1 (H-2s); NIH (H-2s); C57BL/; and Biozzi/ABH.Mice were immunized subcutaneously with the various CVPs in the presenceof QS-21 (10 μg/dose; Aquila Biopharmaceutical Inc., Worcester, Mass.)in a total volume of 100 μl/dose. Either two immunizations on days 0 and21, or three immunizations on days 0, 14 and 28 were administered (seeTable 3, supra). Blood was collected by tail-bleeding or exsanguinationon day 42 as above; sera were collected and stored at −20° C. For thedetection of anti-CPMV antibody, wells were coated with 0.1 μg/well ofCPMV for 3 h at 37° C. A series of doubling dilutions of serum wereincubated on the antigen-coated plates for 1 h at 37° C. Bound antibodywas detected with either alkaline phosphatase (AP)-conjugated goatanti-mouse IgG₁, IgG_(2a), IgG_(2b), or IgG₃ (Southern BiotechnologiesInc., USA) with p-nitrophenyl phosphate (PNPP) (Sigma) as the substrate.

These anti-mouse isotype reagents were first titrated against standardmouse myeloma proteins representing the four IgG subclasses. A dilutionof 1:2000 of each conjugate gives less than 10% variation betweenmyeloma binding. This dilution was used in all subsequent assays.Endpoint titers were calculated as above. For CPMV-specific titers, theresults were expressed as end-point titer, calculated as the inverse ofthe dilution that gives a mean OD₄₀₅ higher than the OD₄₀₅ obtained witha 1:50 dilution of pooled serum from unimmunized mice. Forpeptide-specific titers, end-point titers were the inverse of thedilution that gives a mean OD₄₀₅ higher than the OD₄₀₅ obtained with a1:50 dilution of pooled serum from wild-type CPMV-immunized mice.

All four constructs were shown to elicit high levels of peptide-specificIgG_(2a) relative to levels of IgG₁ or IgG₃ (P<0.01 in all cases),despite the presence of a known TH2 immuno-modulatory adjuvant (that is,QS-21 or alum). Levels of peptide-specific IgG, and IgG₃ werecorrespondingly much lower within all of the immunization groups (Table3, supra). Some of the CVPs also produced significant quantities ofpeptide-specific IgG_(2b). Where this occurred, the levels were lowerthan the levels of IgG_(2a) with the exception of CPMV-MAST1, which insome cases elicited levels of IgG_(2b) that were higher than levels ofIgG_(2a) (Table 3, supra). Thus, CVPs elicited predominantlypeptide-specific TH1-type responses that appear to be influenced byneither the source nor sequence of the expressed peptide nor thepresence of adjuvant with TH2 immunomodulatory potential.

Crucially, however, the CVPs in this experiment elicited TH1-typeresponses in mice of four different H-2 haplotypes. This indicated thatthe effect was not determined or governed by immune response genes (Irgenes). In other words, the genetic disposition of an individual was notan inhibiting factor in the ability of CPMV to elicit a TH1 bias in theimmune response triggered by a particular peptide. It was especiallysignificant that the effect of TH1 bias in an immune response was seenin Biozzi/ABH mice inoculated with CPMV-MAST1. Biozzi/ABH mice aregenetically predisposed to favor a TH2-biased response, even before thepotential biasing effects of extraneous adjuvants or of the peptidesthemselves were taken into account.

Hence, the CPMV-associated TH1 response was capable of overriding strongintrinsic genetic factors generally considered to govern to asignificant extent the immune reaction of an individual. This wasemphasized further by analysis of the specificity of immunoglobulinsfrom TH1 and TH2 pathway sub-types. Less than 1% of IgG1 produced waspeptide-specific, compared with 25% to 100% of total IgG2a in all micein the test group. In summary, the use of CPMV as a carrier andpresentation system for peptides can overcome barriers at the level ofthe genetics of an individual thereby providing a means to obviatevaccine-genomic considerations in the design of prophylactic andtherapeutic agents.

Example 5 Presentation of Peptides on CPMV Particles Induces TH1-BiasedImmune Responses Over a Wide Range of Dosage Regimens

It was apparent from a consideration of the IgG titration data in Table3, supra, that higher levels of peptide-specific IgG_(2a) (indicative ofa TH1-type response) than of peptide-specific IgG₁ (indicative of aTH2-type response) were generated to several different antigensregardless of whether high or low doses (ranging from 300 μg CVPs downto 2 μg) were utilized in the immunization protocol. Thus a furtheradvantage of the immunomodulatory characteristics of CPMV as a carriersystem was the lower amount of material which is required to elicit adesired type of response.

Example 6 CVPs Elicit a TH1-Type Response Whether Presented Parenterallyor Mucosally, Indicating that the Method of Particle Administration DoesNot Affect the Nature of the Immune Response that was Stimulated

NIH mice were immunized intranasally with CPMV-PARVO9, a CVP displayinga peptide derived from the VP2 protein of canine parvovirus. No adjuvantwas used for this inoculation directly onto a mucosal surface (see Table3, row 3). Two doses each containing 100 μg of CVPs were administered ondays 0 and 14. On day 42, blood was collected essentially as describedabove and assayed for the presence of sub-classes of immunoglobulinsspecific for the PARVO9 peptide (Table 3; supra, group 4). Theconcentration of peptide (VP2)-specific antibody was expressed as apercentage of the total antibody for each of the four isotypes withinindividual mice as shown in Table 4. Also, the mean percentage valuesfor each of the four isotypes is shown in Table 4.

TABLE 4 Relative concentration of total and peptide-specific IgGisotypes in CVP-immunized mice Mean total % peptide-specific isotypeMean peptide-specific isotype of total concen- isotype concentrationisotype tration (μg/ml) (μg/ml) concentration IgG₁ 94.5 ± 40.7 <0.004 μg<0.004% IgG_(2a) 135.0 ± 9.7  8.9 ± 3.9 μg 6.6% IgG_(2b) 96.5 ± 17.9 4.7± 3.1 μg 4.9% IgG₃ 44.0 ± 14.3 <0.004 μg <0.004%

Although high levels of total IgG₁ and IgG₃ (which were almostexclusively CPMV specific) were detected, the total concentrations ofIgG_(2a) and IgG_(2b) were also high. Furthermore, significantproportions were peptide-specific (Table 4, supra), highlighting againthe bias in the responses towards the TH1-type and the fact that such aresponse can be elicited regardless of the route of administration ofthe antigen presented on CPMVs.

Example 7 Use of a Chimeric Virus Particle-Containing ImmunogenicComplex to Alter the Nature of an Extant Immune Response

There are situations in which particular antigens are presented asvaccines in the presence of adjuvants which elicit a predominantlyTH2-type response. Indeed, the approval to date of only alum (whichelicits predominantly a TH2-type immune response) as an adjuvant that isconsidered safe for human vaccine applications indicates that manyvaccines that will become available will contain, a priori, a factorwhich elicits a TH2-type immune response with adverse side-effects. Thisemphasizes the need for immunomodulation of a response towards the TH1pathway and away from the TH2 pathway induced in such circumstances.Since many vaccines require multiple inoculations to be effective, it ispossible to address the issue of altering a TH2 response (that isassociated with a given peptide in a subunit vaccine) in favor of aTH1-type response by boosting the initial response with a formulationcontaining a CPMV-presented peptide. The strength of theimmunomodulatory dominance of the CPMV platform bypasses the TH2 pathwaypotential of the adjuvant, while still achieving the required boostingof the immune response to the peptide in question.

To demonstrate this, mice are immunized with MAST1 protein (Table 1) inthe presence of alum and/or QS-21 adjuvants to induce a TH2 response asdetermined by protein-specific IgG levels. Test mice are subsequentlyimmunized with CPMV-MAST1, with control mice receiving no subsequentimmunization. IgG levels in the test and control mice are determined asdescribed supra. An increase in the ratio of peptide-specificIgG1:IgG2a, IgG3:IgG2a, IgG1:IgG2b, and/or IgG3/IgG2b in the testanimals relative to the control animals demonstrates that the chimericviruses of the invention overcome the TH2 response which is induced bythe adjuvant in the primary immunization.

Example 8 The Use of CPMV Virus Particles Engineered to Express andPresent Chemically Reactive Peptides to Conjugate ImmunogenicProteinaceous and Non-Proteinaceous Moieties, Especially CarbohydrateMoieties

Deoxyribonucleotides encoding the amino acid sequence with the formulaDEGKGKGKGKDE (SEQ ID NO:29) are cloned into the vector pCP2corresponding to a cDNA copy of the RNA2 molecule of cowpea mosaicvirus. The insertion is made such that the reactive peptide is insertedinto the βB-βC loop of VP-S (the smaller of the two CPMV virus coatproteins) between alanine 22 and proline 23 as previously described(U.S. Pat. Nos. 5,958,422 and 5,874,087; each is incorporated in itsentirety by reference). Purified carbohydrates are chemically conjugatedto reactive lysine residues in the peptide and the resultingcarbohydrate-conjugated CVPs are purified by affinity-chromatographyusing for example, a carbohydrate-specific antibody or by DEAE-cellulosechromatography. Carbohydrate-conjugated CVPs are inoculated into micefollowing essentially the same immunization regimen as described inExamples 2 and 3 above. Tail bleeds are taken on day 42 and the seraassayed for IgG sub-classes specific for the carbohydrate. Apredominance of TH1 response IgG subclasses is seen consistent with theimmunomodulatory dominance of CPMV.

Example 9 CVPs Prime Predominantly CPMV- and Peptide-Specific IgG_(2a)-and IgG_(2b)-Producing B Cells in Spleen as Determined by ELISPOT

Spleen cells from CPMV-MAST1 (Table 3, row 10) in QS-21 immunized micewere pooled and the red blood cells removed by cold lysis in 0.8% NH₄Cl.The cells were washed twice with RPMI 1640, counted and resuspended atconcentrations of either 5×10⁶, 5×10⁵ or 5×10⁴ viable cells/ml. Eachcell suspension (100 μl/well) was added to the wells of 96-wellMultiscreen Immobilon IP plates (Millipore, Ontario, Canada) which werepre-coated overnight at 4° C. with either CPMV or FnBP peptide insterile carbonate buffer, pH 9.6 and blocked with RPMI 1640 containing10% FCS for one hour at 37° C. Negative control wells were coated onlywith buffer or with an irrelevant control peptide. The cells wereincubated on the plates for 20 h at 37° C. and then washed three timeswith PBS. Bound antibody was detected with the appropriate alkalinephosphatase (AP)-conjugated goat anti-mouse IgG₁, IgG_(2a), IgG_(2b), orIgG₃ (Southern Biotechnologies Inc., USA). After two hours at 37° C.,the plates were washed four times with PBST and streptavidin-peroxidase(100 μl/well) was added for 30 minutes at 37° C. Following washing withPBST, Sigma FAST™ DAB (3,3′-diaminobenzidine tetrahydrochloride)substrate was added (100 μl/well) until maximal color intensitydeveloped. Plates were washed gently with tap water, dried for 30minutes at 37° C., and the spots counted using a dissection microscope(Nikon SMZ-1). The results were expressed as mean spot-forming cells(SFC) per 106 spleen cells (SFC/10⁶ spleen cells) ±SD for both CPMV andpeptide, as shown in Table 5.

TABLE 5 CVPs prime predominantly CPMV and peptide-specific IgG_(2a) andIgG_(2a)-producing B cells in spleen IgG₁ IgG_(2a) IgG_(2b) IgG₃^(a)Mean CPMV - 22200± 666555± 921600± 13000± specific titer ± SD^(b)Mean No.   2 ± 3 240 + 6.8 44 ± 15  0.3 ± 0.8 CPMV - specificSFC/10⁶ spleen cells ± SD ^(a)Mean peptide- 17704± 162696± 197958± 5840± specific titer ± SD ^(b)Mean No. 0.6 ± 1  41 ± 8.3 13 ± 3.7 0.3 ±0.8 peptide-specific SFC/10⁶ spleen cells ± SD ^(a)C57BL/6 mice wereimmunized subcutaneously with CPMV-MAST1 in QS-21 (Table 3, row 10).Sera were collected on day 42 and examined for CPMV- and FnBP-specificIgG₁, IgG_(2a), IgG_(2b) or IgG₃ by ELISA. Titers are expressed asgeometric mean end-point titer ± SD. ^(b)Spleen cells were pooled andexamined for the presence of CPMV- and FnBP-specific IgG₁, IgG_(2a),IgG_(2b) or IgG₃- producing SFC by ELISPOT. The results are expressed asarithmetic mean number of SFC/10⁶ spleen cells ± SD.

ELISPOT analysis of immunoglobulins elicited by CPMV-MAST1 in QS-21showed similarly high numbers of IgG_(2a) and IgG_(2b) spot-formingcells in the spleens of the immunized mice in contrast with the muchlower numbers of IgG₁ and IgG₃ SFCs (Table 5). There were approximately4-fold higher titers of CPMV-specific IgG_(2a) and IgG_(2b) antibody andSFCs compared to peptide (FnBP)-specific antibody and SFC in these mice(Table 5). Interestingly, although levels of FnBP-specific IgG_(2a) andIgG_(2b) in sera were very similar, much higher numbers ofIgG_(2a)-producing SFCs than IgG_(2b) producing SFCs were detected inspleen by ELISPOT (Table 5), suggesting that CPMV-MAST1 primes a largernumber of FnBP-specific IgG_(2a)-producing B cells. Thus, C57BL/6 miceimmunized with CPMV-MAST1 elicited very high concentrations ofpredominantly CPMV-specific IgG_(2a) and IgG_(2b) in serum, with lowerbut significant levels of CPMV-specific IgG₁ and IgG₃ (Table 5).

Example 10 CPMV-Specific T Cells in Spleen Primed by CVPs Proliferate toProduce IFN-γ (a TH1-Associated Cytokine), not IL-4 (a TH2-AssociatedCytokine)

Spleens from mice immunized with CVPs were removed 42 days after primaryimmunization and single cell suspensions were made. Following washingand lysis of red blood cells with ice-cold 0.85% NH₄Cl, the splenocytesfrom five mice were pooled and dispensed into round-bottomed 96-wellplates (2×10⁵/100 μl; Nunclon Delta Surface, Nunc, Denmark) in RPMI 1040medium (Gibco, Paisley, UK) containing 1 mM L-glutamine, 10 mMpenicillin/streptomycin and 10% fetal calf serum (FCS). Cells werecultured with 2.5 μg/ml concanavalin A (ConA, Sigma) or wild-type CPMV(50 or 5 μg/ml) for five days at 37° C. in 5% CO₂. In the last 12 hoursof culture 0.5 μCi/well of (methyl-3H) thymidine (Amersham Life Science)were added. Cells were harvested onto filter mats (Wallac Oy, Turku,Finland) and incorporation of label measured using a 1450 MicrobetaTrilux liquid scintillation and luminescence counter (Wallac). Data wereexpressed as stimulation indices, where counts per minute in thepresence of adjuvant were divided by counts measured in the absence ofantigen (medium only). A value of 3 or greater was consideredsignificant. Spleen cells from mice immunized with CPMV-MAST1proliferated very strongly in vitro to stimulation with even lowconcentrations (5 μg/ml) of CPMV, indicating a highly specific T cellinduction mediated by the peptide carrier.

Pooled spleen cells were subsequently cultured alone or with either ConA(2.5 μg/ml) or wild-type CPMV (50 or 5 μg/ml) in 24-well plates(5×10⁶/well in 2 ml volume). After 48 h, 0.5 ml of cell culturesupernatant were collected into Eppendorf tubes and stored at −80° C.The supernatants were tested for the presence of both IFN-γ and IL-4 byELISA essentially as described above. Briefly, 96-well ELISA plates(Immulon-4) were coated overnight with 2 μg/well of either rat mAbanti-mouse IFN-γ or rat mAb anti-mouse IL-4 (both Pharmingen, San Diego,Calif.) at 4° C. After blocking of the plates with PBS containing 0.05%Tween and 5% BSA, serial dilutions of mouse spleen cell culturesupernatant were added to the wells. As controls, dilutions ofrecombinant mouse IFN-γ and IL-4 (Pharmingen), starting at 4 ng/ml and15 ng/ml for IL-4 and IFN-γ, respectively, were added. After one hour at37° C., bound cytokine was detected using biotinylated rat anti-mouseIFN-γ or anti-IL-4 mAbs (both Pharmingen) for one hour at 37° C. ThesemAbs recognize different epitopes on IFN-γ and IL-4 than the mAbs usedfor the capture step earlier in the procedure. Streptavidin peroxidase(Sigma; 2 μg/ml) was added for 30 minutes at 37° C. followed byO-phenylenediamine (OPD) substrate (1 mg/ml). After 30 minutes at 37°C., the reaction was stopped by the addition of 50 μl/well 2.5 MH₂SO₄,and the absorbance read at 492 nm using an automated Anthos HT II ELISAplate reader. The results are shown in Table 6.

TABLE 6 CPMV stimulates proliferation and cytokine production by spleencells of the TH1 phenotype ^(c)Mean IFN-γ ^(d)Mean IL-4 ^(a)Mean concen-concentra- Antigen CPM ± SD ^(b)SI tration ± SD tion ± SD None  520 ±160 — 0.21 ng/ml 0.04 ng/ml (A) None  520 ± 160 — 0.21 ng/ml 0.04 ng/mlCPMV 13381 ± 2143 25.7 19.24 ng/ml 0.04 ng/ml ConA 11442 ± 1383 22.010.70 ng/ml 0.10 ng/ml (B) None  384 + 184 — NT NT CPMV 316 ± 79 0.820.239 ng/ml 0.04 ng/ml ConA  9333 ± 3559 24.3 NT NT ^(a,b)C57BL/6 miceare immunized subcutaneously with CPMV-MAST1 in QS-21 (Table 3, row 10).Spleen cells from these mice (A) and from unimmunized mice (B) werepooled and cultured for 5 days either alone or with 2.5 μg/ml ConA or 50μg/ml CPMV. T cell proliferation is expressed both as mean CPM ± SD^(a)and SI^(b). ^(c,d)The supernatants from these cultures were alsoexamined for the presence of IFN-γ^(c) and IL-4^(d) protein by ELISA.The results are expressed as arithmetic mean ng/ml ± SD. (NT = nottested).

The supernatants from these proliferating cells were shown to containhigh levels of IFN-γ and low or undetectable levels of IL-4 (Table 6A).Unimmunized mice by comparison contain no CPMV-specific antibody or SFC(not shown) and do not proliferate, nor do they produce IFN-γ or IL-4 inresponse to CPMV stimulation (Table 6B).

Example 11 Conjugation of Peptides

In this example, a chimeric plant virus in accordance with the inventionis engineered to express a reactive peptide which contains a cysteineresidue and which is capable of conjugating with any peptide that hasbeen activated using n-maleimidobenzoyl-N-hydroxysuccinimide ester(“MBS” available from Pierce).

Deoxyribonucleotides encoding the amino acid sequence with the formulaArg-Glu-Arg-Glu-His-Cys (SEQ ID NO:30) are cloned into the vector pCP2corresponding to a cDNA copy of the RNA2 molecule of cowpea mosaicvirus. The insertion is made such that the reactive peptide is insertedinto the βB-βC loop of VP-S (the smaller of the two CPMV virus coatproteins) between alanine 22 and proline 23 as previously described(U.S. Pat. Nos. 5,958,422 and 5,874,087; each is incorporated in itsentirety by reference).

The protein molecule of interest which is sought to be conjugated to theinvention's plant virus is activated using MBS as follows. The proteinis dissolved in buffer (for example, 0.01 M NaPO₄, pH 7.0) to a finalconcentration of approximately 20 mg/ml. At the same time, MBS isdissolved in N,N-dimethyl formamide to a concentration of 5 mg/ml. TheMBS solution, 0.51 ml, is added to 3.25 ml of the protein solution andincubated for 30 minutes at room temperature with stirring every 5minutes. The resulting MBS-activated protein is then purified bychromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume)equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions are pooled(6.0 ml).

The chimeric plant virus expressing Arg-Glu-Arg-Glu-His-Cys (SEQ IDNO:30) (20 mg) is added to the MBS-activated protein solution, stirreduntil the peptide is dissolved and incubated three hours at roomtemperature. Within 20 minutes, the reaction mixture becomes cloudy andprecipitates are formed. After three hours, the reaction mixture iscentrifuged at 10,000×g for 10 minutes and the supernatant analyzed forprotein content. The conjugate precipitate is washed three times withPBS and stored at 4° C. The resulting purified protein-conjugated CVPsare inoculated into mice following essentially the same immunizationregimen as described in Examples 2 and 3 above. Tail bleeds are taken onday 42 and the sera assayed for IgG sub-classes specific for thecarbohydrate. A predominance of TH1 response IgG subclasses is seenconsistent with the immunomodulatory dominance of CPMV.

From the above, it is clear that the invention provides methods andcompositions which are effective in modulating the nature and/or levelof an immune response to any molecule of interest exemplified by, butnot limited to, an antigen or immunogen. In particular, data presentedherein demonstrates that the invention provides methods and means foreffecting a TH1 bias in the immune response to molecules such asantigens or immunogens, and/or reducing a TH2 bias in the immuneresponse to such molecules. More particularly, the above shows thatinvention provides methods and means for increasing a TH1 immuneresponse which is directed against molecules that otherwise generallystimulate a TH2-type response. From the above, it is also evident thatthe invention further provides compositions and methods to reduce a TH2immune response to molecules. The above additionally shows that theinvention furnishes compositions and methods for altering (that is,increasing or decreasing) the level of TH1- and TH2-associatedimmunoglobulins, the level of proliferation of TH1- and TH2-associatedcytokines, and the level of proliferation of TH1 and TH2 cells.

All publications and patents mentioned in the above specification wereherein incorporated by reference. Various modifications and variationsof the described methods and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith a specific preferred embodiment, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiment. Indeed, various modifications of the described modes forcarrying out the invention which were obvious to those skilled in theart and in fields related thereto were intended to be within the scopeof the following claims.

The following useful plant viral vectors are on deposit at the AmericanType Culture Collection (ATCC), Rockville, Md., USA, under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and Regulationsthereunder: pTB2 (ATCC No. 75280) and pTBU5 (ATCC No 75281). Theconstruction details for these plasmids are set forth in U.S. Pat. No.5,589,367 hereby incorporated by reference.

1. A process for producing a virus-like particle: providing a plant virus expressing a heterologous peptide on the exposed portion of the coat protein of said plant virus, which is capable of conjugating to a peptide of interest, wherein said plant virus is a cowpea mosaic virus (CPMV), and wherein said heterologous peptide comprises one or more acidic amino acids and an equal number of basic amino acids; and conjugating said peptide of interest to said heterologous peptide to generate a conjugate.
 2. The process of claim 1, wherein conjugating said peptide of interest comprises chemically conjugating said peptide of interest.
 3. The process of claim 1, wherein said peptide of interest is an antigen of a source selected from the group consisting of an animal pathogen and a cancer cell.
 4. The process of claim 1, wherein said acidic amino acids are selected from aspartic acid, glutamic acid, and cysteine, and said positively charged amino acids are selected from lysine, arginine, and histidine.
 5. The process of claim 1, wherein said heterologous peptide comprises a sequence of contiguous charged amino acids selected from a first sequence consisting of contiguous acidic amino acids and a second sequence consisting of contiguous basic amino acids.
 6. The process of claim 5, wherein said sequences of amino acids occurs in said heterologous peptide as a repeating sequence.
 7. The process of claim 5, wherein said heterologous sequence comprises said first and second sequences, and wherein said first sequence is contiguous with said second sequence.
 8. The process of claim 7, wherein said contiguous first and second sequences occur in said heterologous peptide as a repeating sequence.
 9. The process of claim 1, wherein said heterologous peptide comprises Asp-Glu-Gly-Lys-Gly-Lys-Gly-Lys-Gly-Lys-Asp-Glu listed as SEQ ID NO:29.
 10. The process of claim 1, wherein said conjugate is immunogenic.
 11. A process for producing a virus-like particle: providing a plant virus containing a polynucleotide encoding a heterologous peptide that is expressed on the exposed portion of the coat protein of said plant virus, said heterologous peptide being capable of conjugating to a peptide of interest, wherein said plant virus is a Comovirus, and wherein said heterologous peptide comprises one or more acidic amino acids and an equal number of basic amino acids; infecting a host cell with said plant virus; and allowing replication and expression of said polynucleotide in the host cell.
 12. The process of claim 11, wherein said acidic amino acids are selected from aspartic acid, glutamic acid, and cysteine, and said positively charged amino acids are selected from lysine, arginine, and histidine.
 13. The process of claim 11, wherein said heterologous peptide comprises a sequence of contiguous charged amino acids selected from a first sequence consisting of contiguous acidic amino acids and a second sequence consisting of contiguous basic amino acids.
 14. The process of claim 13, wherein said sequences of amino acids occurs in said heterologous peptide as a repeating sequence.
 15. The process of claim 13, wherein said heterologous sequence comprises said first and second sequences, and wherein said first sequence is contiguous with said second sequence.
 16. The process of claim 15, wherein said contiguous first and second sequences occur in said heterologous peptide as a repeating sequence.
 17. The process of claim 11, wherein said heterologous peptide comprises Asp-Glu-Gly-Lys-Gly-Lys-Gly-Lys-Gly-Lys-Asp-Glu listed as SEQ ID NO:29.
 18. The process of claim 11, wherein said plant virus is a cowpea mosaic virus (CPMV).
 19. A process of increasing the level of an immune response to a peptide of interest in an animal comprising: providing a plant virus expressing a heterologous peptide on the exposed portion of the coat protein of said plant virus, which is capable of conjugating to a molecule of interest, wherein said plant virus is a cowpea mosaic virus (CPMV), and wherein said heterologous peptide comprises one or more acidic amino acids and an equal number of basic amino acids; chemically conjugating said molecule of interest to said heterologous peptide to generate a conjugate; and administering said conjugate to a host animal to generate a treated animal under conditions such that the level of an IgG immunoglobulin response to said molecule of interest in said treated animal is increased relative to the level of said IgG immunoglobulin response to said molecule of interest that is not conjugated to said plant virus in a control animal.
 20. The process of claim 19, wherein the molecule of interest is a protein. 